Viscosity-responsive fluorescent probes, methods of making and using the same

Abstract

The application discloses preparation and application of a viscosity response probe, a viscosity type fluorescent probe and preparation and application thereof, and a structural formula of the fluorescent probe is shown in the following formula: The fluorescent probe prepared by the application has good recognition effect on viscosity.A new multifunctional fluorescent platform CY-RN2 is constructed, which mainly carries out viscosity detection based on a fluorescence resonance energy transfer (FRET) process.The probe has sensitive fluorescence only to viscosity, and the probe has good photochemical stability, near-infrared emission, good solubility and high fluorescence quantum yield.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of chemical technology, in particular to a viscosity-responsive fluorescent probe and a preparation method and application thereof. BACKGROUND

[0002] The interior of a cell is a complex, heterogeneous microenvironment. The cytoplasm contains various organelles and a large number of proteins, nucleic acids, sugars and other biological macromolecules, making the cytoplasmic environment very "crowded". This crowding within the cell not only affects protein folding, enzyme catalysis, intracellular signaling, intracellular material transport and the positioning of molecules and organelles, but also can cause different regions within the cell to have different viscosities. For example, lysosomes in MCF-7 cells are between 50 and 90 cP, while the viscosity values in mitochondria of living HeLa cells are between 60 and 110 cP. It is well known that the viscosity within a cell is an integral part of the biological signaling process, and changes in the viscosity of the microenvironment within a cell can induce various diseases or cause physiological dysfunction. For example, an increase in blood viscosity and abnormal blood rheology characteristics can directly affect tissue perfusion, leading to tissue dehydration and hypoxia, metabolic disorders and muscle dysfunction, causing a series of serious consequences. In addition, the higher the plasma viscosity, the higher the whole blood viscosity. An increase in plasma viscosity in clinical medicine can lead to hereditary spherocytosis, some ischemic cardiovascular diseases, diabetes and macroglobulinemia. Therefore, the viscosity within a cell is considered to be one of the indispensable microenvironment parameters in various biological systems.

[0003] Traditional viscosity detection methods include viscosity meter detection methods, and common viscosity meters include rotary viscometers, glass capillary viscometers, falling ball viscometers, etc. However, traditional viscosity meters can only detect plasma viscosity, and cannot detect cell and tissue samples, nor can they detect microscopic viscosity in vivo. Moreover, there is a large measurement error in the detection process, which can easily contaminate the sample. Compared with traditional detection methods, fluorescence spectroscopy converts chemical and biological signals into detectable signals, and has the characteristics of simple operation, high analysis sensitivity and strong selectivity. And when the fluorescent probe is combined with fluorescence confocal microscopy (FCM), fluorescence lifetime imaging microscopy (FLIM), two-photon imaging microscopy (TPM) and super-resolution fluorescence microscopy (SRM), the fluorescent probe can be more easily applied to the detection of related molecules in vivo and the analysis of related physiological and pathological phenomena. In recent years, with the rapid development of microscopic imaging technology, a large number of fluorescent probes have been developed for labeling and detecting various important analytes in the environment and biology, achieving real-time monitoring of cells and tissues and playing an important role in the field of biology.

[0004] The sensing mechanism of these fluorescent probes mainly relies on the change of fluorescence after interacting with target species through some known sensing mechanisms, such as intramolecular charge transfer (ICT), photoinduced electron transfer (PET), aggregation-induced emission (AIE), and decomposition-induced emission (DIE). Most of these probes have a unique chemical target by intelligently selecting the sensing and recognition mode. Fluorescent molecular rotors have been widely used to monitor the changes of cell viscosity, and they play a key role in understanding the function of viscosity-related diseases. SUMMARY

[0005] Based on the technical problems existing in the background art, the present application provides a preparation method of a viscosity fluorescent probe and its application, which is sensitive to viscosity fluorescence only, and has good photochemical stability, near-infrared emission, good solubility and high fluorescence quantum yield.

[0006] We constructed a new multifunctional fluorescent platform CY-RN2, which mainly detects viscosity based on the fluorescence resonance energy transfer (FRET) process. The probe can better detect the average viscosity in cells.

[0007] The molecular formula of the viscosity-responsive fluorescent probe provided by the present application is C 102 H 102 ClN 10 O6 3+ ·Cl2O8I 3- , and the structural formula is:

[0008] .

[0009] The preparation method of the viscosity-responsive fluorescent probe provided by the present application comprises the following steps:

[0010] S1: 20 mL N-N dimethylformamide is added to a flask, and then phosphorus trichloride is added dropwise, followed by slow addition of cyclohexanone for reaction. After the reaction is completed, pour into the prepared ice water, and there is a yellow precipitate. Filter, wash, dry, and finally obtain compound 1.

[0011] S2: 2,2,3-trimethylbenzoindole and 5-chloro-1-pentynyl, acetonitrile are respectively placed in 50 mL three-necked flasks and potassium iodide, and reacted at 85℃ under nitrogen protection for 24 h, filtered, rotary dried solvent, and the residue is recrystallized with acetone to obtain compound 2.

[0012] S3: Compound 2 is dissolved in n-butanol / benzene, compound 1 is added, reacted, and then cooled to room temperature, and rotary dried under reduced pressure. The product is purified by thin layer chromatography to obtain compound 3.

[0013] S4: Under ice bath, cyclohexanone was added into 20 mL concentrated sulfuric acid, and 2-(4-diethylamino-hydroxybenzoyl)benzoic acid was added under vigorous stirring. After the dropwise addition was completed, the reaction was condensed and refluxed under nitrogen protection. After the reaction was completed, it was cooled to room temperature, poured into ice, 2.0 mL perchloric acid was added, and a large amount of solid was precipitated. Finally, the solid was removed by filtration, washed with cold water for 3 times, and dried in air to obtain a red solid The red solid was reacted with p-azidobenzaldehyde in acetic acid to obtain compound 4.

[0014] S5: Compound 3 and compound 4 were dissolved in 20 mL dimethyl sulfoxide, then sodium ascorbate was added, and finally 2.0 mL copper sulfate aqueous solution was added, and the reaction was carried out at 35 ℃ overnight under nitrogen protection. After the reaction was completed, it was cooled to room temperature, extracted with water and dichloromethane for 3 times, and the solvent was evaporated under reduced pressure. The product was purified by thin layer chromatography to obtain black compound 6 (probe CY-RN2).

[0015] Preferably, the molar ratio of phosphorus trichloride to cyclohexanone in S1 is 2:1.

[0016] Preferably, the molar ratio of 2,2,3-trimethylbenzoindole, 5-chloro-1-pentynyl and potassium iodide in S2 is 1:1:2.

[0017] Preferably, the molar ratio of compound 2 to compound 1 in S3 is 2:1, and the volume ratio of n-butanol to benzene is 7:3.

[0018] Preferably, the molar ratio of cyclohexanone to 2-(4-diethylamino-hydroxybenzoyl)benzoic acid in S4 is 2:1. The molar ratio of the red solid to p-azidobenzaldehyde is 1:1.

[0019] Preferably, the molar ratio of compound 3 to compound 4 in S5 is 1:2.

[0020] The viscosity-responsive fluorescent probe prepared by the above method of the present application.

[0021] The viscosity-responsive fluorescent probe prepared by the above method of the present application.

[0022] The detection steps of the above application are as follows:

[0023] (1) Preparation of solution: 1 mmol / mL probe solution was prepared by using dimethyl sulfoxide solution as solvent, and viscosity solution was prepared by using deionized water and glycerol to prepare solutions with different volume ratios;

[0024] (2) Fluorescence spectrum test: F-7100 fluorescence spectrophotometer was used, 2 mL solvent and 20 μL probe solution were added into a quartz dish, mixed to make the concentration constant at 10 μmol / mL, and scanning was carried out with 620 nm as the excitation wavelength.

[0025] Beneficial effects:

[0026] The present application constructs a new multifunctional fluorescence platform CY-RN2, which mainly detects viscosity based on fluorescence resonance energy transfer (FRET) process. The probe has sensitive fluorescence only to viscosity, and the probe has good photochemical stability, near-infrared emission, good solubility and high fluorescence quantum yield. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 The synthesis route of the viscosity-responsive fluorescence probe proposed in the present application is shown in the figure;

[0028] Figure 2 The mass spectrum of CY-RN2 in the viscosity-responsive fluorescence probe proposed in the present application is shown in the figure;

[0029] Figure 3 The ultraviolet absorption graph of the viscosity-responsive fluorescence probe CY-RN2 proposed in the present application is shown in the figure;

[0030] Figure 4 The linear relationship graph of the fluorescence intensity (732 nm and 820 nm) of CY-RN2 and different volume ratio viscosity solutions proposed in the present application is shown in the figure;

[0031] Figure 5 The fluorescence spectrum of the probe CY-RN2 in the presence of different ions in an aqueous phase proposed in the present application is shown in the figure. DETAILED DESCRIPTION

[0032] The present application will be further described below in combination with specific embodiments.

[0033] Example 1

[0034] The method steps for preparing the viscosity-responsive fluorescence probe proposed in the present application are as follows:

[0035] S1: 20 ml N-N dimethylformamide (DMF) was added to a flask, and phosphorus trichloride 110 mmol was added dropwise, reacted for 30 min, and then cyclohexanone 50 mmol was slowly added to carry out the above reaction. After the reaction was completed, pour into prepared ice water, filter out the solid, wash with water for several times, dry the obtained solid under vacuum, and finally obtain light yellow solid 1.

[0036] S2: 2,2,3-trimethylbenzoindole 10 mmol and 5-chloro-1-pentynyl 10 mmol, 8 mL acetonitrile were placed in a three-necked flask and potassium iodide 22 mmol respectively, and reacted at 85°C for 24 h under nitrogen protection. After filtration, the solvent was rotary evaporated, and the residue was recrystallized with acetone to obtain compound 2.

[0037] S3: 2 mmol of compound 2 was dissolved in 80 mL of n-butanol / benzene (7 / 3, v / v), and 1 mmol of compound 1 was added. After condensation refluxing at 135°C for 6 h under nitrogen protection, the reaction was cooled to room temperature, and the product was rotary evaporated under reduced pressure. The product was purified by thin layer chromatography (dichloromethane:methanol = 10:1, v / v) to obtain compound 3.

[0038] S4: Cyclohexanone 20 mmol was added dropwise to 20 mL of concentrated sulfuric acid under ice bath, and 2-(4-diethylamino-hydroxybenzoyl)benzoic acid 10.0 mmol was added under vigorous stirring. After dropwise addition, condensation refluxing was carried out at 90°C for 3 h under nitrogen protection. After the reaction was completed, it was poured into 150 g of ice, and (2.0 mL, 70%) perchloric acid was added, and a large amount of solid was precipitated. After filtration, washing, and drying in air, a red solid was obtained . The red solid 0.376 g and p-azidobenzaldehyde 0.147 g were added to 10 mL of acetic acid, and reacted at 90°C for 12 h under nitrogen protection to obtain compound 4.

[0039] S5: 10 mmol of compound 3 and 20 mmol of compound 4 were dissolved in 60 mL of dimethyl sulfoxide, 40 mg of sodium ascorbate was added, and finally 10 mL of copper sulfate aqueous solution was added. After condensation refluxing at 35°C overnight under nitrogen protection, the reaction was cooled to room temperature, extracted with water and dichloromethane, and the solvent was evaporated under reduced pressure. The product was purified by thin layer chromatography (dichloromethane:methanol = 2:1, v / v) to obtain compound 6 (probe CY-RN2).

[0040] Example 2

[0041] The viscosity fluorescent probe prepared in the present embodiment was structurally characterized and its performance was studied, and the results are as follows:

[0042] 1. Fluorescent sensing of viscosity by probe CY-RN2

[0043] I. Fluorescence spectrum test

[0044] (1) Preparation of solution: 1 mmol / mL of probe solution was prepared using dimethyl sulfoxide solution as solvent, and viscosity solutions were prepared by using deionized water and glycerol in different volume ratios;

[0045] (2) Fluorescence spectrum test: F-7100 fluorescence spectrophotometer was used, 2 mL solvent and 20 μl probe solution were added in a quartz dish, mixed to make the concentration constant at 10 μmol / mL, and scanning was performed with 620 nm as the excitation wavelength. The emission peak of CY-RN2 was at 732 nm and 820 nm, and the fluorescence spectrum was drawn by Origin.

[0046] Ⅱ Fluorescence sensing of probe to viscosity

[0047] As shown in Figure 4, when the probe concentration in the aqueous phase was 10 μmol / mL, the excitation wavelength was 620 nm, and the probe CY-RN2 had a relatively weak emission peak at 732 nm and 820 nm. However, as we changed the viscosity in the aqueous phase, we found that as the viscosity increased, the fluorescence intensity at 732 nm and 820 nm increased significantly (a), and there was a good linear relationship between the fluorescence intensity and the viscosity at this wavelength (b, 4c). Figure 4 log[I 732 ] and log[viscosity], log[I 820 ] and log[viscosity] had a correlation coefficient of 0.99312 and 0.99406, respectively. Figure 4

[0048] The probe had weak or almost no response to other ions and amino acids (Ca 2+ , Cu 2+ , Fe 3+ , Fe 2+ , H2O2, S2O8 2- , I - , S 2- , S2O3 2- , SO4 2- , ClO - , NO 2- , NH 4+ , Pb 2+ , Zn 2+ , Ag + , SO3 2- , Cl2O7 2- , Cys, Leu, Trp, Thr) (d). Figure 5 ​​

Claims

1. A viscosity fluorescent probe, characterized in that, The probe structure is as follows: 。 2. A method for preparing the viscosity fluorescent probe according to claim 1, characterized in that, Includes the following steps: 。 3. The application of the viscosity fluorescent probe of claim 1 in viscosity detection for purposes other than disease diagnosis and treatment.

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