阅读说明：本技术 一种异恶唑类化合物作为粘度荧光探针的应用 (Application of isoxazole compound as viscosity fluorescent probe ) 是由 王宇光 陈圆 刘贝 于 2021-08-12 设计创作，主要内容包括：本发明提供一类检测细胞内粘度变化的荧光探针分子,该探针合成路径简便,以水为反应溶剂,减少了有机溶剂的使用,响应可持续发展理念,实现了溶剂的零排放。反应中巧妙地使用了TPGS-750-M在水中形成微胶束来解决有机化合物在水中的溶解性问题,充分利用了水的优秀理化性质,反应条件温和,反应高效。并且表面活性剂TPGS-750-M通过处理可以回收利用,完全符合环境友好的原则。该探针对粘度变化敏感,能实现溶液中粘度的检测,并且可以实现细胞内粘度检测与成像,在荧光生物标记领域也有潜在的应用价值。(The invention provides fluorescent probe molecules for detecting viscosity change in cells, the synthesis path of the probe is simple and convenient, water is used as a reaction solvent, the use of an organic solvent is reduced, the sustainable development concept is responded, and zero emission of the solvent is realized. In the reaction, TPGS-750-M is skillfully used to form micro-micelles in water so as to solve the problem of solubility of organic compounds in water, and the excellent physicochemical properties of water are fully utilized, so that the reaction condition is mild, and the reaction is efficient. And the surfactant TPGS-750-M can be recycled through treatment, and completely accords with the environment-friendly principle. The probe is sensitive to viscosity change, can realize detection of viscosity in a solution, can realize intracellular viscosity detection and imaging, and has potential application value in the field of fluorescence biomarker.)

1. The application of the isoxazole compound shown in the formula (III) as a viscosity fluorescent probe,

2. the use of claim 1, wherein: the application is the application of the isoxazole compound shown in the formula (III) as a viscosity fluorescent probe in the detection of the viscosity of living cells.

3. The use according to claim 2, characterized in that the use is: after the living cells are incubated in a medium containing the isoxazole compound represented by the formula (III), the viscosity is compared by fluorescence detection.

4. Use according to claim 3, characterized in that: the concentration of the isoxazole compound of formula (III) in the medium of the isoxazole compound of formula (III) is 5. mu.M.

5. Use according to claim 3, characterized in that: the incubation conditions were 37 ℃ for 20 min.

6. The use according to claim 3, wherein the conditions for fluorescence detection are: detection was performed using confocal imaging at an excitation wavelength of 410 nm.

Technical Field

The invention belongs to the field of organic small-molecule fluorescent probes, and particularly relates to a viscosity-sensitive isoxazole fluorescent probe synthesized in a water phase and biological application thereof in intracellular viscosity detection.

Background

Isoxazoles are a common structural scaffold in many biologically active molecules. Isoxazole derivatives also have a wide range of biological activities, such as neurotoxin (A), antidepressant-like active substance (B), anti-beta-lactamase antibiotic (C), and synthetic androgen danazol (D) with histone deacetylase (HADC) probe (E) which inhibits gonadotropin production. In addition, isoxazole derivatives can also be used as main precursors for synthesizing different organic compounds.

Viscosity, as one of the main parameters affecting biological processes, largely determines the mobility of substances and the reaction rate of diffusion-controlled reactions. Intracellular viscosity abnormalities are important indicators of dysfunction at the cellular level in many diseases, such as common hypertension, diabetes, and alzheimer's disease, among others. The fluorescent probe has high sensitivity, good specificity and simple operation, so the fluorescent probe with viscosity sensitivity can be used as an important tool for measuring the intracellular viscosity.

Based on the technical current situation, the invention aims to provide an isoxazole fluorescent probe sensitive to viscosity. The probe is sensitive to the change of viscosity, the fluorescence intensity of the probe is increased along with the increase of viscosity concentration, and the probe can be well applied to the detection of intracellular viscosity.

Disclosure of Invention

The invention aims to provide application of an isoxazole compound as a viscosity fluorescent probe. The probe is sensitive to the viscosity change of the solution, and the fluorescence intensity of the probe is obviously enhanced along with the increase of the viscosity of the solution; the probe has no toxicity to cells basically, and can be used for viscosity detection and imaging in living cells.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides application of an isoxazole compound shown as a formula (III) as a viscosity fluorescent probe, which is referred to as probe I for short.

The invention particularly recommends the application of the isoxazole compound shown in the formula (III) as a viscosity fluorescent probe in the viscosity detection of living cells.

Further, the application is as follows: after the living cells are incubated in a medium containing the isoxazole compound represented by the formula (III), the viscosity is compared by fluorescence detection.

Preferably, the medium for the isoxazole compound of formula (III) has a concentration of the isoxazole compound of formula (III) of 5. mu.M.

Further, the incubation condition was 37 ℃ for 20 min.

Further preferably, the conditions for fluorescence detection are: detection was performed using confocal imaging at an excitation wavelength of 410 nm.

In the present invention, the isoxazole compound of the formula (III) is prepared by the following method:

the preparation method comprises the following steps:

s1: sequentially adding N-chlorosuccinimide (NCS) and benzaldehyde oxime shown in a formula (I) into a reaction bottle, then adding a surfactant into a reaction system, and stirring the mixed solution at room temperature for 1-4 hours;

s2, adding a certain amount of alkali and 4-dimethyl amino phenylacetylene shown in the formula (II) into the reaction system in sequence, and reacting for 4-12 hours at room temperature.

S3: after the reaction is finished, adding ethyl acetate into the reaction bottle, stirring for five minutes, separating and draining an organic phase to obtain:

the preparation method can be further improved on the basis of the technical scheme as follows.

Further, 4-dimethylaminophenylacetylene represented by the formula (II) was used as a standard, and the amount of NCS used was 1.8 times equivalent to that of 4-dimethylaminophenylacetylene;

further, the base used in the reaction is selected from one of the following: et (Et)₃N、K₂CO₃Piperidine, based on 4-dimethylaminophenylacetylene of formula (II), preferably 1.3 equivalents of triethylamine;

further, the surfactant used in the reaction is selected from one of the following: SDS, TPGS-750-M, TritonX-100, preferably 2 wt.% TPGS-750-M.

Reacting the substance (II) in the reaction processExchange for substance (IV)Then the second probe (V) can be preparedThe reaction formula is as follows:

the isoxazole fluorescent probe prepared by the invention is sensitive to viscosity change and has low cytotoxicity, and can be used for viscosity detection in living cells.

The surfactants used in the invention, namely Sodium Dodecyl Sulfate (SDS) and polyethylene glycol octyl phenyl ether (Trition X-100), are commercially available products, and the tocopherol methoxypolyethylene glycol succinic acid (TPGS-750-M, water-soluble vitamin E) is self-made, preferably 2 wt.% of TPGS-750-M;

further preferably, the TPGS-750-M is prepared as follows:

(1) triethylamine was added to a toluene solution of tocopherol and succinic anhydride with stirring. Stirring was then continued. After completion of the reaction, water was added to the reaction mixture, followed by extraction with dichloromethane. The combined organic layers were washed with 1mol/L hydrochloric acid and water, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure in vacuo to give a yellow liquid, which was purified using silica gel column chromatography. Removing the solvent from the eluent under vacuum reduced pressure to obtain tocopherol succinate in a white solid state;

(2) the toluene mixture containing tocopherol succinate, polyethylene glycol monomethyl ether-750 and p-toluenesulfonic acid was refluxed using a dean-Stark trap. After the reaction was completed, it was cooled to room temperature, and the mixture was poured into a saturated aqueous sodium hydrogencarbonate solution and extracted with dichloromethane. The combined organic layers were washed with saturated sodium bicarbonate and saturated sodium chloride, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure in vacuo to give the desired product.

Compared with the prior art, the invention has the beneficial effects that: the viscosity probe provided by the invention is a fluorescent probe molecule for detecting viscosity change in cells, the synthesis path of the probe is simple, water is used as a reaction solvent, the use of an organic solvent is reduced, the sustainable development concept is responded, and zero emission of the solvent is realized. In the reaction, TPGS-750-M is skillfully used to form micro-micelles in water so as to solve the problem of solubility of organic compounds in water, and the excellent physicochemical properties of water are fully utilized, so that the reaction condition is mild, and the reaction is efficient. And the surfactant TPGS-750-M can be recycled through treatment, and completely accords with the environment-friendly principle. The probe is sensitive to viscosity change, can realize detection of viscosity in a solution, can realize intracellular viscosity detection and imaging, and has potential application value in the field of fluorescence biomarker.

Drawings

FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of a first viscosity fluorescent probe;

FIG. 2 is a nuclear magnetic resonance carbon spectrum of a first viscosity fluorescent probe;

FIG. 3 is a nuclear magnetic resonance hydrogen spectrum of a viscosity fluorescent probe II;

FIG. 4 is a nuclear magnetic resonance carbon spectrum of a viscosity fluorescent probe II;

FIG. 5 is a fluorescence emission spectrum of a viscosity fluorescence probe-fluorescence intensity with PBS/glycerol solvents of different viscosities;

FIG. 6 is a linear plot of the logarithm of the fluorescence intensity at the wavelength of maximum absorption versus the logarithm of the viscosity, where the abscissa is the logarithm of the viscosity and the ordinate is the logarithm of the fluorescence intensity;

FIG. 7 is a fluorescence emission spectrum of a viscosity fluorescence probe-fluorescence intensity with PBS/glycerol solvents of different viscosities;

FIG. 8 is a fluorescence emission spectrum of a selective result of the viscosity fluorescent probe I under PBS/glycerol conditions. 1-24 are respectively (1) blank; (2) mn²⁺；(3)Ser；(4)Phe；(5)Arg；(6)Cys；(6)BSA；(7)H₂S； (8)GSH；(9)Hcy；(10)CO₃ ^2-；(11)OAc^-；(12)NO^3-；(13)HSO₃ ^-；(14)PO₄ ^3-；(15)Cl^-； (16)Fe³⁺；(17)K⁺；(18)ClO^-；(19)H₂O₂；(20)ONOO^-；(21)Zn²⁺；(22)Cu²⁺；(23)Li⁺； (24)Glyceral；

FIG. 9 is a cytotoxicity study of different concentrations of first viscosity fluorescent probes;

FIG. 10 is a confocal fluorescence image of viscosity in HeLa cells containing 5 μ M probe one and different viscosity concentrations: (a, d) fluorescence images of hela cells loaded with 5mM nystatin, respectively, with an excitation wavelength of 410 nm; (b, e) bright field of Hela cells loaded with 5mM nystatin, respectively. Emission was collected between 450 and 500 nm; (c, f) mixed channels of Hela cells loaded with 5mM nystatin, respectively. Emission was collected between 450 and 500 nm.

FIG. 11 shows TPGS-750-M¹H NMR spectrum;

FIG. 12 shows TPGS-750-M¹³C NMR spectrum.

Detailed Description

The present invention is further illustrated below with reference to examples, which are by no means intended to limit the scope of the invention.

The surfactant Sodium Dodecyl Sulfate (SDS) and the polyethylene glycol octyl phenyl ether (Trition X-100) used in the invention are commercially available products, and the tocopherol methoxypolyethylene glycol succinic acid solution (TPGS-750-M, water-soluble vitamin E) is self-made, and preferably 2 wt.% of TPGS-750-M.

The surfactant TPGS-750-M used in the examples was self-made and the preparation method was as follows:

first step of

Second step of

The first step is as follows: to a stirred solution of tocopherol (4.3g, 10mmol) and succinic anhydride (1.5g, 15mmol) in toluene (20mL) was added triethylamine (0.35mL, 2.5 mmol). Subsequently, stirring was continued at 60 ℃ for 5 h. After completion of the reaction, water (10mL) was added to the reaction mixture, followed by extraction with dichloromethane (3X 10 mL). The combined organic layers were washed with 1mol/L hydrochloric acid (3X 50mL) and water (2X 30mL), dried over anhydrous sodium sulfate, and concentrated in vacuo to give crude tocopherol succinate, which was finally recrystallized to give tocopherol succinate as a white solid (5.25g, 99% yield). The crystallization conditions were: the crystallization time is 26 hours, the crystallization temperature is 4 ℃, and the dosage of n-hexane/tocopherol succinate crude product is 8 mL/g.

The second step is that: a mixture of tocopherol succinate (2.97g, 5.6mmol), polyethylene glycol monomethyl ether-750 (4g, 5.33mmol) and p-toluenesulfonic acid (0.15g, 0.79mmol) in toluene (20mL) was refluxed using a dean-Stark trap for 5 hours. After the reaction was completed, it was cooled to room temperature, and the mixture was poured into a saturated aqueous sodium hydrogencarbonate solution and extracted with dichloromethane (3X 10 mL). The combined organic layers were washed with saturated sodium bicarbonate (3 × 50mL), saturated sodium chloride (2 × 30mL), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure in vacuo to give the title product (6.6g, 98%) in 80% yield as a pale yellow waxy solid.

¹H NMR(400MHz,CDCl₃)δ4.27(m,2H),3.70-3.65(m,60H),3.55(m,2H), 3.38(s,3H),2.93(t,J＝7.2Hz,2H),2.79(t,J＝7.2Hz,2H),2.58(t,J＝6.8Hz,2H), 2.08(s,3H),2.01(s,3H),1.97(s,3H),1.81-1.75(m,2H),1.52(m,3H),1.38(m,3H), 1.27-1.23(m,12H),1.14(s,3H),1.07(s,3H),0.87-0.84(m,12H)；¹³C NMR(100 MHz,CDCl₃) δ 172.2,170.9,149.5,140.6,126.7,125.0,123.0,117.4, 75.1,72.0, 70.6,70.58-70.33(m, carbons), 69.1,64.0,59.0,39.4,37.55-37.29(m, carbons), 32.79-32.69(m,4C),29.2,28.9,28.0,24.816,24.805,24.5,22.8,22.7,21.1,20.6, 19.77-19.61(m, carbons), 113.0, 12.1,11.8.

Example 1: synthesis of Probe N, N-dimethyl-4- (3-phenyl-5-isoxazolyl) aniline (III)

A50 mL reaction flask was charged with 241mg (1.8mmol) NCS, 182mg (1.5mmol) benzaldoxime (formula I), then 10mL of freshly prepared 2 wt.% TPGS-750-M aqueous solution was added to the flask, and after stirring the mixture at room temperature for 4 hours, 182. mu.L (1.3mmol) Et was added sequentially₃N, 145mg (0.1mmol) of 4- (N, N-dimethylamino) phenylacetylene (II), reacting for 8 hours at room temperature, adding 15mL of ethyl acetate into a reaction bottle, stirring for 5 minutes, standing for layering, collecting an upper organic phase, adding 15mL of ethyl acetate into a lower aqueous phase, stirring for 5 minutes, standing for layering, collecting an upper organic phase, extracting TPGS-750-M in the lower aqueous phase with dichloromethane to obtain separation and recovery (see example 6), combining the two obtained organic phases, removing the solvent by suspension evaporation under reduced pressure, and separating solid residues by column chromatography, wherein an eluent is petroleum ether: ethyl acetate (v/v, same below) 1: 2, collecting the eluent containing the target product, and evaporating the solvent under reduced pressure to obtain 260.9mg of N, N-dimethyl-4- (3-phenyl-5-isoxazolyl) aniline shown in the formula (III) as a white solid with the yield of 96.5%. The purity is 98.7%.

¹H NMR(600MHz,CDCl₃)δ7.89(dd,J＝7.9,1.4Hz,2H),7.73(d,J＝8.9Hz, 2H),7.51-7.45(m,3H),6.78(d,J＝8.9Hz,2H),6.65(s,1H),3.06(s,6H)；¹³C NMR (126MHz,CDCl₃)δ171.25,162.84,151.44,129.75,129.62,128.84,127.11,126.81, 111.87,94.66,40.21GC-MS(EI):m/z 264[M⁺].

Example 2: synthesis of Probe N, N-dimethyl-4- (3-phenyl-5-isoxazolyl) aniline (III)

A5 mL reaction flask was charged with 24.1mg (0.18mmol) NCS, 18.2mg (0.15mmol) benzaldoxime (formula I), then 1mL of freshly prepared 2 wt.% TPGS-750-M aqueous solution was added to the flask, and after stirring the mixture at room temperature for 4 hours, 7. mu.L (0.05mmol) Et was added sequentially₃N, 14.5mg (0.1mmol) of 4-dimethylaminophenylacetylene are reacted at room temperature for 8 hours. After the reaction is finished, adding 2mL ethyl acetate into a reaction bottle, stirring for 5min, standing for layering, collecting an upper organic phase, adding 2mL ethyl acetate into a lower aqueous phase, stirring for 5min, standing for layering, collecting an upper organic phase, extracting TPGS-750-M in the lower aqueous phase with dichloromethane to obtain separation and recovery (see example 6), combining the two obtained organic phases, removing the solvent by suspension evaporation under reduced pressure, and separating solid residues by column chromatography, wherein an eluent is petroleum ether: ethyl acetate ═ 1: 2, collecting the eluent containing the target product, and evaporating the solvent under reduced pressure to obtain 16.6mg of N, N-dimethyl-4- (3-phenyl-5-isoxazolyl) aniline shown in the formula (III) as a white solid with the yield of 63%.

Example 3: synthesis of Probe N, N-dimethyl-4- (3-phenyl-5-isoxazolyl) aniline (III)

To a 5mL reaction flask was added 24.1mg (0.18mmol) NCS, 18.2mg (0.15mmol) benzaldoxime (formula I), then 1mL of freshly prepared 2 wt.% TPGS-750-M aqueous solution was added to the flask and the mixture was stirred at room temperature for 4 hours. After four hours, 21. mu.L (0.15mmol) of Et were added successively₃N, 14.5mg (0.1mmol) of 4-dimethylaminophenylacetylene are reacted at room temperature for 8 hours. After the reaction, 2mL of ethyl acetate is added into the reaction bottle, the mixture is stirred for 5min and then is kept stand for layering, after an upper organic phase is collected, 2mL of ethyl acetate is added into a lower aqueous phase, the mixture is kept stand for layering after being stirred for 5min, after the upper organic phase is collected, TPGS-750-M in the lower aqueous phase is extracted by dichloromethane to obtain separation and recovery (see example 6), the organic phases obtained in two times are combined, and the suspension evaporation is carried out under reduced pressure to remove TPGS-750-M in the lower aqueous phaseSeparating the solvent and the solid residue by column chromatography, wherein the eluent is petroleum ether: ethyl acetate ═ 1: 2, collecting the eluent containing the target product, and evaporating the solvent under reduced pressure to obtain 24.5mg of N, N-dimethyl-4- (3-phenyl-5-isoxazolyl) aniline shown in the formula (III) as a white solid with the yield of 93%.

Example 4: synthesis of Probe N, N-dimethyl-4- (3-phenyl-5-isoxazolyl) aniline (III)

A5 mL reaction flask was charged with 24.1mg (0.18mmol) NCS, 18.2mg (0.15mmol) benzaldoxime (formula I), then 1mL of freshly prepared 2 wt.% TPGS-750-M aqueous solution was added to the flask, and after stirring the mixture at room temperature for 4 hours, 28. mu.L (0.2mmol) Et was added sequentially₃N, 14.5mg (0.1mmol) of 4-N' N dimethylaminophenylacetylene are reacted at room temperature for 8 hours. After the reaction is finished, adding 2mL ethyl acetate into a reaction bottle, stirring for 5min, standing for layering, collecting an upper organic phase, adding 2mL ethyl acetate into a lower aqueous phase, stirring for 5min, standing for layering, collecting an upper organic phase, extracting TPGS-750-M in the lower aqueous phase with dichloromethane to obtain separation and recovery (see example 6), combining the two obtained organic phases, removing the solvent by suspension evaporation under reduced pressure, and separating solid residues by column chromatography, wherein an eluent is petroleum ether: ethyl acetate ═ 1: 2, collecting the eluent containing the target product, and evaporating the solvent under reduced pressure to obtain 23.7mg of N, N-dimethyl-4- (3-phenyl-5-isoxazolyl) aniline shown in the formula (III) as a white solid with the yield of 90%.

Example 5: synthesis of Probe 3, 5-Diphenylisoxazole (V)

To a 50mL reaction flask was added 241mg (1.8mmol) NCS, 182mg (1.5mmol) benzaldoxime (I), then 10mL of freshly prepared 2 wt.% aqueous TPGS-750-M solution was added to the flask and the mixture was stirred at room temperature for 4 hours. After four hours, 168. mu.L (1.2mmol) of Et were added successively₃N, 14.5mg (0.1mmol) of 4-N' N dimethylaminophenylacetylene (IV) are reacted at room temperature for 8 h. After the reaction, 15mL of ethyl acetate was added to the reaction flask, and the mixture was stirredStirring for 5min, standing for layering, collecting the upper organic phase, adding 15mL ethyl acetate into the lower aqueous phase, stirring for 5min, standing for layering, collecting the upper organic phase, extracting TPGS-750-M in the lower aqueous phase with dichloromethane to obtain a separated and recovered solution (see example 6), combining the two organic phases, performing suspension evaporation under reduced pressure to remove the solvent, and performing column chromatography separation on the solid residue, wherein the eluent is petroleum ether: ethyl acetate ═ 1: 2, collecting the eluent containing the target product, and evaporating the solvent under reduced pressure to obtain 214.6mg of 3, 5-diphenylisoxazole shown in the formula (V) as a white solid with the yield of 97%. The structure of formula (V) is characterized as follows:

¹H NMR(600MHz,CDCl₃)δ7.86(dd,J＝17.0,7.9Hz,4H),7.53-7.43(m,6H), 6.84(s,1H)；¹³C NMR(151MHz,CDCl₃)δ170.44,163.01,130.26,130.05,129.15, 129.04,128.96,127.49,126.84,125.87,97.50；GC-MS(EI):m/z 221[M⁺].

example 6: synthesis of Probe N, N-dimethyl-4- (3-phenyl-5-isoxazolyl) aniline (III)

To a 500mL reaction flask was added 2.41g (18mmol) NCS, 1.82g (15mmol) benzaldoxime (I), then 100mL of freshly prepared 2 wt.% aqueous TPGS-750-M solution was added to the flask and the mixture was stirred at room temperature for 4.5 hours. Then, 1.82mL (13mmol) of Et was added₃N, 1.45g (10mmol) of phenylacetylene (II) were reacted at room temperature for 8 hours. After the reaction is finished, adding 50mL of ethyl acetate into a reaction bottle, stirring for 5min, standing for layering, collecting an upper ethyl acetate phase, adding 50mL of ethyl acetate into a lower water phase, stirring for 5min, standing for layering, collecting an upper ethyl acetate phase, extracting TPGS-750-M in the lower water phase twice with 50mL of dichloromethane respectively, combining dichloromethane extraction solutions, evaporating dichloromethane under reduced pressure, and recovering to obtain 1.6g of TPGS-750-M; and (3) combining the ethyl acetate phases obtained in the two steps, evaporating the solvent under reduced pressure, and separating solid residues by column chromatography, wherein the eluent is petroleum ether: ethyl acetate ═ 1: 2, collecting the eluent containing the target product, and evaporating the solvent under reduced pressure to obtain 2.564g of N, N-dimethyl-4- (3-phenyl-5-isoxazolyl) aniline shown in the formula (III) as a white solid with the yield of 97%. Purity by HPLC 98.8%.

Example 7: synthesis of Probe N, N-dimethyl-4- (3-phenyl-5-isoxazolyl) aniline (III)

To a 5mL reaction flask was added 24.1mg (0.18mmol) NCS, 18.2mg (0.15mmol) benzaldoxime (formula I), then 1mL of freshly prepared 2 wt.% TPGS-750-M aqueous solution was added to the flask and the mixture was stirred at room temperature for 4 hours. After four hours, 13.8mg (0.1mmol) of K are added₂CO₃14.5mg (0.1mmol) of 4-dimethylaminophenylacetylene are reacted at room temperature for 8 hours. After the reaction is finished, adding 2mL ethyl acetate into a reaction bottle, stirring for 5min, standing for layering, collecting an upper ethyl acetate phase, adding 2mL ethyl acetate into a lower water phase, stirring for 5min, standing for layering, collecting an upper ethyl acetate phase, combining the two obtained ethyl acetate phases, evaporating the solvent under reduced pressure, separating the residue by column chromatography, wherein the eluent is petroleum ether: ethyl acetate ═ 1: 2, collecting the eluent containing the target product, and evaporating the solvent under reduced pressure to obtain 8.5mg of N, N-dimethyl-4- (3-phenyl-5-isoxazolyl) aniline shown in the formula (III) as a white solid with the yield of 32%.

Example 8: synthesis of Probe N, N-dimethyl-4- (3-phenyl-5-isoxazolyl) aniline (III)

To a 5mL reaction flask was added 24.1mg (0.18mmol) NCS, 18.2mg (0.15mmol) benzaldoxime (formula I), then 1mL of freshly prepared 2 wt.% TPGS-750-M aqueous solution was added to the flask and the mixture was stirred at room temperature for 4 hours. After four hours, 10.2. mu.L (0.1mmol) of piperidine and 14.5mg (0.1mmol) of 4-dimethylaminophenylacetylene were added in succession and reacted at room temperature for 8 hours. After the reaction is finished, adding 2mL ethyl acetate into a reaction bottle, stirring for 5min, standing for layering, collecting an upper ethyl acetate phase, adding 2mL ethyl acetate into a lower water phase, stirring for 5min, standing for layering, collecting an upper ethyl acetate phase, combining the two obtained ethyl acetate phases, evaporating the solvent under reduced pressure, separating the residue by column chromatography, wherein the eluent is petroleum ether: ethyl acetate ═ 1: 2, collecting the eluate containing the target product, and evaporating the solvent under reduced pressure to obtain 18.2mg of N, N-dimethyl-4- (3-phenyl-5-isoxazolyl) aniline represented by the formula (III) as a white solid with a yield of 69%.

Example 9: synthesis of Probe N, N-dimethyl-4- (3-phenyl-5-isoxazolyl) aniline (III)

To a 5mL reaction flask was added 24.1mg (0.18mmol) NCS, 18.2mg (0.15mmol) benzaldoxime (formula I), then 1mL of freshly prepared 2 wt.% TPGS-750-M aqueous solution was added to the flask and the mixture was stirred at room temperature for 4 hours. After four hours, a further 14. mu.L (0.1mmol) of Et are added₃N, 14.5mg (0.1mmol) of 4-dimethylaminophenylacetylene are reacted at room temperature for 8 hours. After the reaction is finished, adding 2mL ethyl acetate into a reaction bottle, stirring for 5min, standing for layering, collecting an upper ethyl acetate phase, adding 2mL ethyl acetate into a lower water phase, stirring for 5min, standing for layering, collecting an upper ethyl acetate phase, combining the two obtained ethyl acetate phases, evaporating the solvent under reduced pressure, separating the residue by column chromatography, wherein the eluent is petroleum ether: ethyl acetate ═ 1: 2, collecting the eluent containing the target product, and evaporating the solvent under reduced pressure to obtain 24.8mg of N, N-dimethyl-4- (3-phenyl-5-isoxazolyl) aniline shown in the formula (III) as a white solid with the yield of 94%.

Example 10: synthesis of Probe N, N-dimethyl-4- (3-phenyl-5-isoxazolyl) aniline (III)

In a 5mL reaction flask were added 24.1mg (0.18mmol) NCS, 18.2mg (0.15mmol) benzaldoxime (formula I), then 1mL SDS (15 mol%) was added to the flask, and the mixture was stirred at room temperature for 4 hours. After four hours, a further 14. mu.L (0.1mmol) of Et are added₃N, 14.5mg (0.1mmol) of 4-dimethylaminophenylacetylene are reacted at room temperature for 8 hours. After the reaction, 3mL of ethyl acetate was added to the reaction flask, and after stirring for 5min, the organic phase was separated and drained to obtain 14.5mg of N, N-dimethyl-4- (3-phenyl-5-isoxazolyl) aniline represented by the formula (III) as a white solid with a yield of 55%.

Example 11: synthesis of Probe N, N-dimethyl-4- (3-phenyl-5-isoxazolyl) aniline (III)

To a 5mL reaction flask were added 24.1mg (0.18mmol) NCS, 18.2mg (0.15mmol) benzaldoxime (formula I), then 1mL Triton X-100 was added to the flask, and the mixture was stirred at room temperature for 4 hours. After four hours, a further 14. mu.L (0.1mmol) of Et are added₃N, 14.5mg (0.1mmol) of 4-dimethylaminophenylacetylene are reacted at room temperature for 8 hours. Inverse directionAfter the reaction, 3mL of ethyl acetate was added to the reaction flask, and after stirring for 5min, the organic phase was separated and drained to obtain 15.8mg of N, N-dimethyl-4- (3-phenyl-5-isoxazolyl) aniline represented by the formula (III) as a white solid with a yield of 60%.

Example 12: sensitivity of Probe one (III) to viscosity

(1) Materials and methods

An amount of probe was weighed and dissolved in dimethylsulfoxide to make a 5mM stock solution, and all spectroscopic measurements were performed in Phosphate Buffered Saline (PBS) in the experiment.

Viscosity solutions were prepared by mixing PBS and glycerol in different ratios. PBS-glycerol solutions of different viscosities at a final concentration of probe one of 5.0. mu.M were prepared by adding stock solutions of probe one (5.0mM, 10.0. mu.L) to PBS-glycerol solutions (10.0mL) mixed at different ratios, respectively. These solutions were sonicated for 10 minutes to eliminate air bubbles and allowed to stand at constant temperature for 1 h. Then, a fluorescence spectrum was recorded under excitation at 410nm, with excitation and emission slit widths set at 5 nm. The relationship between the fluorescence emission intensity of the probe and the viscosity of the solvent is well expressed by the Forster-Hoffmann equation as follows:

lg(If)＝k lgη+c

where If is the fluorescence intensity, η is the viscosity of the solution, and k and c are constants.

(2) Preparation of PBS-Glycerol buffers of different viscosities

Respectively putting 10mL, 20mL, 30mL, 40 mL, 50mL, 60 mL, 70 mL, 80 mL, 90 mL and 95mL of glycerol into a 100mL volumetric flask, slowly adding a PBS solution into the volumetric flask along a glass rod, dripping by using a rubber head dropper when the distance is about 1 cm from a marked line, finally enabling the meniscus of the liquid to be just tangent to the marked line, and standing for 1h at normal temperature (25 ℃) for later use.

(3) Sensitivity of Probe one and Probe two to viscosity

The first probe and the second probe were dissolved in dimethyl sulfoxide to prepare 5mM solutions. mu.L of the above-mentioned stock solutions of probe one and probe two were added to 10mL of PBS-glycerin solutions having different viscosities (prepared in example 12, (2)), and the mixture was allowed to stand at room temperature (25 ℃) for 30min and then divided into two portions. One of the samples was measured for viscosity using a viscometer (Shanghai Pingxuan scientific Instrument Co., Ltd., NDJ-8S), and the other was used for fluorescence detection.

The viscosities of the PBS (0.01% DMSO, pH 7.4) -glycerol solutions after probe one addition were: 99.56cp, 198.76cp, 290.12cp, 386.01cp, 482.66cp, 579.25cp, 676.36cp, 769.58cp, 863.95cp and 954.52 cp; another part is added into a 96-well plate respectively, fluorescence detection is carried out at the excitation wavelength of 410nm, as can be seen from FIG. 5, the fluorescence intensity of the probe I is gradually enhanced along with the increase of the viscosity of the system, and the logarithm of the fluorescence intensity of the probe and the logarithm of the viscosity show a good linear relation in the viscosity range of 99.56cp to 954.52cp (FIG. 6), and the linear formula is: 1.39347x-1.17522, linear relation coefficient R²0.99598. This shows that the probe can respond well to viscosity in glycerol system.

The viscosities of the PBS (0.01% DMSO, pH 7.4) -glycerol solutions after probe two addition were: 99.58cp, 198.81cp, 290.19cp, 386.09cp, 482.72cp, 579.35cp, 676.47cp, 769.67cp, 864.01cp and 954.59 cp; fluorescence measurements were performed at an excitation wavelength of 410nm, with fluorescence intensities as shown in FIG. 7, and the viscosities and their corresponding fluorescence intensities at the absorption maxima are given in the following table:

from the above table and fig. 7, it can be seen that the fluorescence intensity of probe two gradually increases with the increase of the viscosity of the system, but the increase of the fluorescence intensity is not significant enough (from 50.31 to 129.37) compared with probe one, i.e. the response relationship of the probe two to the viscosity in the glycerol system is not significant. The increase of the fluorescence intensity of probe one is very obvious (from 38.15 to 965.46) and the logarithm of the fluorescence intensity at the maximum absorption wavelength thereof shows a good linear relationship with the logarithm of the viscosity, and the probe one has a good response relationship with the viscosity in a glycerol system.

Example 13: selective study of Probe one

Selecting ROSCommon substances in (active oxygen cluster), RCS (active carbon cluster), biological thiol and metal ions are taken as analytes for selectivity test, and specifically comprises (1) Blank; (2) mn2 +; (3) ser; (4) Phe; (5) arg; (6) cys; (6) BSA; (7) H2S; (8) a GSH; (9) hcy; (10) CO 32-; (11) OAc-; (12) NO 3-; (13) HSO 3-; (14) PO 43-; (15) cl-; (16) fe3 +; (17) k +; (18) ClO-; (19) H2O 2; (20) ONOO-; (21) zn2 +; (22) cu₂(ii) a (23) Li +; (24) the analytes were each made up as a 50 mM stock solution in water. To 10mL of 5. mu.M probe-one PBS buffer (0.1% DMSO, pH 7.4) was added 10. mu.L of the biologically-relevant analyte, respectively. Standing at normal temperature for 30min, and performing fluorescence detection. The result is shown in FIG. 8, from which it can be seen that at an excitation wavelength of 410nm, other substances cause substantially no significant fluorescence spectrum change, except for a slight fluorescence enhancement caused by viscosity, demonstrating that the probe has a very good specificity for viscosity at an emission wavelength of 460 nm.

Example 14: cytotoxicity study of Probe one

The toxicity of the probes to hela cells was tested using the MTT method. Hela cells were cultured on 96-well cell culture plates (1X 10)⁴Individual cells/well), at 37 ℃ cell culture chamber (containing 5% CO)₂) And (4) incubating for 24 hours, wherein the culture medium is high-sugar DMEM culture medium with 10% fetal calf serum and proper resistance (100U/ml penicillin and 100ug/ml streptomycin). Probe one (0-50. mu.M) was then added thereto and incubated for 24 h. 50 μ L of MTT was added to each well and cells were incubated at 37 ℃ in 5% CO₂The mixture was incubated for 4 hours. The medium was then removed and DMSO (150. mu.L) was added to each well and absorbance was measured at 410 nm. As can be seen from FIG. 9, the probe is substantially non-toxic to Hela cells and is suitable for viscosity detection and imaging in cells.

Example 15: probe one cell imaging

Hela cells were cultured on 96-well cell culture plates (1X 10)⁴Individual cells/well), at 37 ℃ cell culture chamber (containing 5% CO)₂) And (5) incubating for 24 hours, wherein the culture medium is a DMEM culture medium with 10% fetal calf serum and high glucose. Dividing the HeLa cells into experimental group and control group, adding into the culture solution of the cells of the experimental groupThe final concentration of mycotin was 5. mu.M, and the incubation of the cells in the experimental group with this medium was continued for 30min, while the control group did not. Thereafter, the first probe was added to the culture medium of the Hela cells of the experimental group and the control group so that the final concentration of the first probe in the culture medium was 5. mu.M, and the incubation with the culture medium was continued for 20 min. Then, the cells were imaged on a confocal imaging dish using a confocal microscope using an excitation wavelength of 410 nm. The results show that the cells show only negligible fluorescence in the absence of nystatin (fig. 10a, c). Significant yellow fluorescence was observed for cells treated with 5mM nystatin (fig. 10d, f). These data show that the probe is capable of performing viscosity measurements in live hela cells using fluorescence methods.