CN102617467A

CN102617467A - Ultrahigh-sensitivity fluorescent probe for detecting nitrogen monoxide

Info

Publication number: CN102617467A
Application number: CN2012100386366A
Authority: CN
Inventors: 肖义; 于海波; 张玉慧; 张晓琳
Original assignee: Dalian University of Technology
Current assignee: Dalian University of Technology
Priority date: 2012-02-21
Filing date: 2012-02-21
Publication date: 2012-08-01

Abstract

The invention relates to an ultrahigh-sensitivity fluorescent probe for detecting nitrogen monoxide. The ultrahigh-sensitivity fluorescent probe has the structural general formula in the original text. During preparation, firstly, 4-bromine-1, 8-anhydride naphthalene and o-nitro p-phenylenediamine with equal molar weights are used as raw materials, dissolved in ethylene glycol monomethyl ether and heated for reaction to obtain light yellow solid. Secondly, the light yellow solid is dissolved in anhydrous DMSO, primary amine is added into the anhydrous DMSO, the anhydrous DMSO is heated and refluxed, and reaction liquid is filtered in a sucking manner, depressurized and dried to obtain yellow solid. The yellow solid is dissolved in mixed liquid of tetrahydrofuran and methanol, and a yellow probe is obtained under catalysis of hydrogen and catalysts. Probe molecules have fine chemical and optical stability, good solubility and biological compatibility and high nitric oxide selectivity, and are free of interference of other active oxygen, active nitrogen and the like. Laser confocal imaging experiments indicate that the probe has fine cellular permeability, has no toxic and side effects on cells and organisms, and can be used for detecting protogenetic nitrogen monoxide in a high-sensitivity manner.

Description

The nitric oxide production ultra-high sensitive fluorescent probe of a kind of detection

Technical field

The present invention relates to one type and detect nitric oxide production highly sensitive naphthalimide fluorescent probe.

Background technology

Nitrogen protoxide is played an important role in life science, is an important messenger molecule.From the eighties in 20th century, scientists is found in the organism vital movement, can produce nitric oxide molecule.Nitrogen protoxide not only has the physiological functions such as inhibition of immunomodulatory, neurotransmission, blood pressure physiological regulating control and platelet aggregation, but also with a lot of diseases, comprise that diseases such as transgenation, canceration, arteriosclerosis are closely related.Yet; Its growing amount of the nitrogen protoxide that produces in the organism is less and the transformation period is shorter; To its carry out in real time, original position, quantitative detection; To help us further to understand nitric oxide production physiological function, and help to understand the pathogenesis of some disease, and finally find suitable treat-ment.

At present nitric oxide production detection method is mainly contained electrochemical process, colourimetry, mass spectroscopy, paramagnetic method.In order to realize the nitrogen protoxide that produces in the biological sample is carried out the detection of real-time in-situ; Utilize the fluorescence detection of fluorescent probe to show special advantages in recent years: fluorescent probe is low to organism toxicity; Do not damage sample cell, through with the routine monitoring instrument--fluorescent microscope combines nitrogen protoxide is realized the detection of original position.The fluorophore of at present nitrogen protoxide being discerned mainly contains resorcinolphthalein, rhodamine, BODPIY, cyanine type dye etc.; Wherein resorcinolphthalein DAF-2 series and rhodamine DAR-2 series are the nitrogen protoxide detection fluorescent probes of present stapleization (InvitrogenTM); Yet its synthesis and preparation process is more loaded down with trivial details; Cause its cost higher, cost an arm and a leg, be unfavorable for extensively promoting the use of.

Although at present the NO production in the life entity and the mechanism of action have been carried out corresponding research exploration, yet, because the NO production amount is less, the life-span is shorter, and only in specific organoid, produce.Can better understand the effect of nitrogen protoxide in vital movement to its detection of carrying out original position, with and as the regulating and controlling effect of messenger molecule to biomacromolecule, therefore be badly in need of that design is low-cost, highly sensitive nitric oxide fluorescent probe.Especially in recent years the development of fluorescence imaging microtechnique, and seldom, and report as yet in the world based on the nitric oxide fluorescent probe of naphthalimide fluorescent chromophore.To the detection of nitric oxide production real-time in-situ, be badly in need of research, develop highly sensitive nitrogen protoxide probe for ease with practicality.

Summary of the invention

The invention provides one type and detect nitric oxide production naphthalimide fluorescent probe.Its general structure is shown in (I):

Two of the object of the invention provides following method and realizes:

The compound method of this naphthalimide fluorescent probe is carried out as follows:

A, with the 4-bromo-1 of equimolar amount, 8-naphthalene acid anhydride and adjacent nitro p-phenylenediamine are raw material, are dissolved in the ethylene glycol monomethyl ether, are heated to 120 ℃, react and after 5 hours reaction solution poured in the water, suction filtration, drying under reduced pressure obtains faint yellow solid.

B, the resulting faint yellow solid of a step is dissolved among the anhydrous DMSO (DMSO 99.8MIN.), adds the substituted primary amine of faint yellow solid equimolar amount, reflux 5 hours is poured reaction solution in the water into, suction filtration, and drying under reduced pressure obtains yellow solid.Primary amine can be third, butylamine, diglycolamine, 2-(N-methyl) piperazine ethamine, 2-morpholine ethamine, 2-amino-ethyl triphenyl phosphorus or 3-aminopropyl triphenyl phosphorus.

C, the resulting yellow solid of b step is dissolved in the mixed solution of THF and methyl alcohol, under the hydrogen and catalyst of 2.5MPa, stirring at room 24 hours obtains the fluorescent probe product.THF: methyl alcohol=5: 1, catalyzer are Pd/C, Raney's nickel, Fe/HCl or tin protochloride.

The preparation feedback formula of above-mentioned naphthalimide fluorescent probe is following:

Naphthalimide fluorescent probe of the present invention is used for the chemical simulation living things system and detects nitric oxide molecule, and nitric oxide production analyzing and testing in viable cell and the tissue slice and fluorescence imaging analysis.Viable cell is Hella cell strain, MCF-7 cell strain, RAW264.7 cell strain etc.

The characteristic that naphthalimide fluorescent probe of the present invention has is following:

The excitation wavelength of this probe is in the 400-460nm scope, and is insensitive to solvent polarity, and chemicalstability is good; Probe molecule has higher selectivity to nitrogen protoxide, other bioactive molecules such as H ₂O ₂, singlet oxygen, peroxynitrite root etc. are noiseless to it, and have higher sensitivity, its sensitivity is in the 0.5-2nmol scope; Through selecting dissimilar primary amine for use, so that regulate the solvability and the pH of probe, to improve its solvability and cell permeability, this fluorescent probe pair cell itself and organism have no side effect, and are applicable to nitric oxide production detection in cell and the biological tissue.

Significant contribution of the present invention is to have synthesized the original nitric oxide production naphthalimide fluorescent probe of a series of detections; It has the interference that long emission wavelength (530nm) can be avoided the autofluorescence of cell or tissue; Improve imaging depth, reduce light injury biological tissue; The Design Mechanism of middle probe of the present invention is based on photoinduced electron transport mechanism, and reasonably regulates group, has realized primary nitrogen protoxide in highly selective, the highly sensitive detection cell; Through the cell imaging experiment, all there is nitrogen protoxide in observing of success in plastosome and the lysosome, and this provides directly perceived and simple and direct biological detection reagent for nitric oxide production pathological research.

Description of drawings

Fig. 1 is the response diagram of fluorescent probe among the present invention (embodiment 5) to pH.

Fig. 2 be probe (embodiment 5) in chemical environment to nitric oxide production response diagram.

Fig. 3 is the laser co-focusing imaging that probe (embodiment 5) is used to study MCF-7 cell intracellular nitric oxide.

Fig. 4 is that probe (embodiment 5) is used for studying the nitric oxide production laser co-focusing imaging of lysosome in the RAW264.7 cell.

Embodiment

Embodiment 1

A is according to 4-bromo-1, and 8-naphthalene acid anhydride and adjacent nitro p-phenylenediamine's equimolar amount proportioning are dissolved in respectively in the ethylene glycol monomethyl ether, stir following 120 ℃ of back flow reaction 5 hours, and reaction solution is poured in the frozen water, and suction filtration obtains faint yellow solid, productive rate 90%, HRMS410.9855

The product that b obtains a operation is dissolved among the DMSO, adds the propylamine of equimolar amount, and 120 ℃ were reacted 5 hours, and reaction solution is poured in the frozen water, and suction filtration obtains yellow solid, productive rate 85%, and HRMS 390.1325

The product that c obtains the b operation is dissolved in the mixed solvent of THF and methyl alcohol (volume ratio is 5: 1), adds 10%Pd/C as catalyzer, the H of 2.5MPa ₂, stirring at room 24 hours with the reactant evaporated under reduced pressure, obtains yellow solid after reaction finishes, productive rate 90%, and HRMS 360.1576

Embodiment 2

Replace propylamine with butylamine in the b operation, all the other are with instance 1, productive rate 92%HRMS 374.1733

Embodiment 3

Use N in the b operation, N-dimethylamino quadrol replaces propylamine, and all the other are with instance 1, productive rate 91%, and HRMS 389.1850

Embodiment 4

Replace propylamine with 2-(N-methyl) piperazine ethylamine in the b operation, all the other are with instance 1, productive rate 87%, HRMS444.2730

Embodiment 5

Replace propylamine with 2-morpholine ethylamine in the b operation, all the other are with instance 1, productive rate 85%, and HRMS 431.1950

Embodiment 6

Replace propylamine with two sweet amine in the b operation, all the other are with instance 1, productive rate 93%, and HRMS 406.1640

Embodiment 7

Replace propylamine with amine ethyl triphenyl phosphorus in the b operation, all the other are with instance 1, productive rate 78%, and HRMS 607.2252

Embodiment 8

Replace propylamine with amine propyl group triphenyl phosphorus in the b operation, all the other are with instance 1, productive rate 80%, and HRMS 621.2410.

Claims

1. one kind is detected nitric oxide production ultra-high sensitive fluorescent probe, it is characterized in that this fluorescent probe has following general structure:

In the formula:

2. the compound method of the said ultra-high sensitive fluorescent probe of claim 1, its characteristic comprises the steps:

A, with the 4-bromo-1 of equimolar amount, 8-naphthalene acid anhydride and adjacent nitro p-phenylenediamine are raw material, are dissolved in the ethylene glycol monomethyl ether, are heated to 120 ℃, react and after 5 hours reaction solution poured in the water, suction filtration, drying under reduced pressure obtains faint yellow solid;

B, the faint yellow solid that step a is obtained are dissolved among the anhydrous DMSO (DMSO 99.8MIN.), add the primary amine of faint yellow solid equimolar amount, and reflux 5 hours is poured reaction solution in the water into, suction filtration, and drying under reduced pressure obtains yellow solid;

C, the yellow solid that step b is obtained are dissolved in the mixed solution of THF and methyl alcohol, and under the hydrogen and catalyst of 2.5MPa, stirring at room 24 hours obtains the fluorescent probe product; THF: methyl alcohol=5: 1; Catalyzer is Pd/C, Raney's nickel, Fe powder/Hydrogen chloride or tin protochloride.

3. compound method as claimed in claim 2; It is characterized in that; Described primary amine is propylamine, butylamine, diglycolamine, N-methyl-2-piperazine quadrol, 2-morpholine quadrol, N, N-dimethyl-ethylenediamine, 2-amino-ethyl triphenyl phosphorus or 3-aminopropyl triphenyl phosphorus.

4. application rights requires 1 described ultra-high sensitive fluorescent probe, it is characterized in that, is used for the nitric oxide production detection of chemical simulation physiology system or is used for the nitric oxide production detection of viable cell.

5. application as claimed in claim 4 is characterized in that, said viable cell is Hella cell strain, MCF-7 cell strain, RAW264.7 cell strain.