Viscosity and so2 dual-responsive fluorescent probe, and preparation method and application thereof

Abstract

The application provides a viscosity and SO2 dual-response fluorescent probe, a preparation method and application thereof, and belongs to the technical field of organic small-molecule fluorescent probes.The fluorescent probe of the application takes barbituric acid and derivatives as an electron donor (Donor, D), and 3-quinoline formaldehyde iodonium salt as an electron acceptor (Acceptor, A) to construct a fluorescent molecular probe with a D-2A pi system based on Knoevenagel condensation reaction.The probe realizes high selectivity to viscosity by using a twisted intramolecular charge transfer effect; after a 1,4-Michael addition reaction occurs between the 4-position of the quinoline iodonium salt and SO2, the quinoline iodonium salt is converted from an electron acceptor to an electron donor, thereby causing a fluorescence signal change to achieve the purpose of detecting SO2, and the viscosity response signal and the SO2 response signal do not cross.The fluorescent probe of the application has the advantages of high sensitivity, low interference and low toxicity, and can be used for detecting viscosity and SO2 in actual samples such as saccharides, lake water and food viscous agents, and has a wide application prospect in the field of biomolecule detection and actual samples.

Description

TECHNICAL FIELD

[0001] The present application relates to a kind of preparation method and application of fluorescent probe for detecting viscosity and SO2, belong to organic small molecule fluorescent probe field. BACKGROUND

[0002] Cell microenvironment is closely related to cell metabolism, proliferation and differentiation, and is an extremely important factor for determining the physical or chemical behavior of surrounding molecules. As a basic physical parameter, viscosity affects diffusion in biological processes. Changes in intracellular viscosity lead to the occurrence of related diseases. In cell biology and tumor pathology, monitoring the changes in cell viscosity is of great significance for identifying abnormal cells. In addition to impairing normal function, abnormal increase in the viscosity of damaged cells can also lead to atherosclerosis, abnormal blood pressure, diabetes, Alzheimer's disease and Parkinson's disease. Studies have found that the viscosity of platelets and red blood cells in diabetic patients is higher than that in normal people. Therefore, accurate monitoring of the viscosity changes in various regions of biological cells and understanding the relationship between disease pathogenesis and intracellular viscosity changes are beneficial for the diagnosis and treatment of related diseases.

[0003] SO2 is mainly present in the form of bisulfite (HSO3 - ) and sulfite (SO3 2- ) after dissolving in water. Sulfite has antibacterial and fresh-keeping properties and is an indispensable additive in food storage. At the same time, sulfite participates in the oxidation-reduction balance of the human body, cell signal transmission, physiological and pathological processes. Excessive intake of sulfite can cause some common respiratory diseases, cardiovascular diseases and white matter encephalopathy. Considering these potential health threats, many countries have strictly limited the content of sulfite to within 0-0.7 mg / kg of the daily intake of the human body. Therefore, it is of great significance to develop a rapid, effective and highly selective method for real-time detection of sulfite.

[0004] In recent years, fluorescence labeling imaging technology combined with fluorescent probes has been widely used due to its high sensitivity, real-time spatial resolution imaging, low light damage to living cells or tissues and low background interference. This technology is one of the important tools for monitoring biological molecules and physiological metabolic processes in living cells and living organisms. Currently, the reported fluorescent probes are mainly for single detection of viscosity or SO2, and there are few fluorescent probes for simultaneous detection of viscosity and SO2. The dual-function fluorescent probes reported for detecting viscosity and SO2 have the problems of spectral overlap, low detection sensitivity, low signal-to-noise ratio and short emission wavelength.

[0005] Based on the above requirements, a kind of near infrared emission fluorescent probe for simultaneously detecting viscosity and SO2 is developed, and it has the characteristics of small degree of spectral cross for detecting two substances, high signal-to-noise ratio and large stokes shift, which has very important scientific significance and practical value for meeting the detection of small molecules in vivo, live body fluorescent imaging labeling and practical application. SUMMARY

[0006] The purpose of the present application is to provide a kind of dual-function fluorescent probe for detecting viscosity and sulfite and its preparation method, to solve the problems of single detection of viscosity and sulfite, spectral cross and small stokes shift of current fluorescent probe.

[0007] The technical scheme adopted by the present application is:

[0008] The fluorescent probe can be used for simultaneously detecting viscosity and sulfite, and the structure of the fluorescent probe is shown as (I):

[0009]

[0010] In formula (I), R is one of methyl and ethyl; X is one of S and O;

[0011]

[0012] When R is methyl and X is O, the structure is shown as (II);

[0013] When R is ethyl and X is S, the structure is shown as (III);

[0014] The present application also provides a preparation method of the above fluorescent probe, and the preparation process is as follows:

[0015]

[0016] The specific preparation steps are as follows:

[0017] S1, 2, 6-dimethylpyranone and barbituric acid or derivative are added to a round-bottom flask containing acetic anhydride, heated under nitrogen or argon protection condition, cooled, filtered, and yellow powdery solid is obtained, which is recrystallized with ethanol to obtain yellow crystalline powder A;

[0018] S2, in a round-bottom flask, compound A and 3-quinoline formaldehyde are added, ethanol is used as reaction solvent and piperidine is used as acid binding agent, the reaction is sealed and oscillated, after the reaction is stopped, the solid is filtered out, and then the solid is ultrasonically treated with acetonitrile to obtain orange solid B;

[0019] S3, compound B and iodomethane are added into the Schlenk tube, and the reaction is carried out in the dark after sealing, after the reaction is completed, fluoroboric acid is added, and after stirring at room temperature for 2 hours, the obtained solid is extracted with acetonitrile and diethyl ether, and ultrasonic treatment is performed to obtain a red solid, which is the target probe molecule shown in formula (I).

[0020] As an embodiment of the present application, in S1, the condensation reaction is carried out for 5 hours at 120 DEG C; in S2, the aldehyde methylation reaction is carried out for 24-48 hours at 90 DEG C; and in S3, the nitrogen methylation reaction is carried out for 24 hours at 60 DEG C.

[0021] As an embodiment of the present application, in S1, the feeding ratio of 2,6-dimethylpyranone and barbituric acid or derivatives is 1:1-2; in S2, the feeding ratio of compound A and 3-quinoline aldehyde in the condensation reaction is 1:2-2.5; the volume of piperidine added is 2 ‰; and in S3, the feeding ratio of compound B and iodomethane in the nitrogen methylation reaction is 1:15-25.

[0022] As an embodiment of the present application, in S1, compound A is obtained by recrystallization with ethanol and then extracted; in S2, compound B is obtained by adding piperidine as the most basic acid; and in S3, the target compound is obtained by adding fluoroboric acid and stirring at room temperature.

[0023] Further, the present application also provides the application of the above-mentioned viscosity and SO2 dual-response fluorescent probe, which is used for detecting SO2 and viscosity in food, water sample and food thickening agent, or is made into test paper to detect the concentration of SO3 in liquid. 2-

[0024] The detection mechanism of the fluorescent probe of the present application is as follows: for viscosity: in a low-viscosity solvent, the carbon-carbon single bond of the fluorescent probe can rotate freely, allowing the probe to return to the ground state as a non-radiative transition from the excited state, resulting in the attenuation of fluorescence emission. On the contrary, in a high-viscosity solvent, the rotation ability between the carbon-carbon single bonds of the probe is inhibited, so that the probe molecule exists in a planar molecular conformation, and the range of the conjugated system formed by the probe molecule is expanded, thereby resulting in a significant enhancement of the fluorescence intensity and quantum yield of the probe. For SO2: HSO3 - / SO3 2- can attack the 4-position of the quinoline iodonium salt of the probe to occur 1,4-Michael addition reaction, so that the quinoline iodonium salt is converted from an electron-withdrawing group to an electron-donating group, and a clear fluorescence signal is generated.

[0025] The present application has at least the following beneficial effects:

[0026] ​(1) The fluorescent probe (named BBA-gamma-Q and TBA-gamma-Q respectively) of the present application is used for specific recognition of viscosity, and the maximum emission wavelength of the probe of the present application in glycerol solution is 582 nm and 605 nm respectively. The fluorescence intensity under the maximum emission wavelength of the probe presents a good linear relationship (R 2 >0.99) with viscosity gradient. Moreover, the probe of the present application has good optical stability, fast response, good biological membrane permeability and low cytotoxicity; the intracellular viscosity can be detected and the change of viscosity in the ferroptosis process can be tracked.

[0027] (2) The fluorescent probe (named BBA-gamma-Q and TBA-gamma-Q respectively) of the present application has high selectivity for SO3 2- , and has the characteristics of near-infrared emission wavelength, selective specificity, fast response, high detection sensitivity and the like. After the fluorescent probe of the present application reacts with SO3 2- , the maximum emission wavelength is located at 761 nm and 772 nm respectively, and the fluorescence intensity under the maximum emission wavelength presents a good linear relationship (R 2- =0.99) with the concentration of SO3 2 , and the detection limit reaches 0.85 nM.

[0028] (3) The fluorescent probe of the present application can be used for detection of SO2 and viscosity in food (white sugar, crystal sugar), water sample (tap water, drinking water and Tianlihu water) and food thickening agent (sodium carboxymethyl cellulose, pectin, gelatin, gum arabic, xanthan gum, sodium alginate and guar gum). Meanwhile, the fluorescent probe can be made into test paper to detect the concentration of SO3 2- in liquid. The preparation method provided by the present application has low cost, high yield and obvious economic and technical effects.

[0029] (4) The fluorescent probe of the present application emits light at 582 nm and 605 nm after reacting with viscosity, and emits light at 761 nm and 772 nm after reacting with SO3 2- . The emission bands of the two detection analytes are low in overlap, which is beneficial to biological imaging and actual sample detection. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 is the fluorescence emission diagram of the probe BBA-gamma-Q prepared in Example 1 of the present application in different viscosity systems;

[0031] Figure 2 is the SO3 2- fluorescence titration experiment of the probe BBA-gamma-Q prepared in Example 1 of the present application;

[0032] Figure 3 is the normalized spectrum of the probe BBA-gamma-Q prepared in Example 1 of the present application responding to viscosity and sulfite.

[0033] Figure 4 Probe BBA-γ-Q prepared in Example 1 of the present application was used to prepare test strips for detecting SO3 2- Figure;

[0034] Figure 5 Probe BBA-γ-Q prepared in Example 1 of the present application was used to prepare test strips for detecting SO3 DETAILED DESCRIPTION

[0035] It should be noted that the following detailed description is illustrative only, and is intended to provide further description in order to provide a fuller enabling teaching of the present application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0036] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, steps, operations, elements, components, and / or groups thereof, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof.

[0037] The technical solutions of the present application will be described in detail below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative labor fall within the protection scope of the present application.

[0038] In order to better understand the technical solutions of the present application, the following will be further described in detail through specific embodiments:

[0039] The reagents used are all of analytical purity, and are directly purchased from reagent companies such as Inokai. Nuclear magnetic resonance spectrum is determined by Bruker spectrometer 400 (MHz); mass spectrum is determined by Agilent 6510Q-TOF LC / MS instrument (Agilent Technologies, Palo Alto, CA), and fluorescence spectrum is determined by Hitachi F-4600 spectrofluorophotometer;

[0040] Example 1: Synthesis of probe BBA-γ-Q

[0041]

[0042] To a 100 mL round bottom flask, 2,6-dimethylpyranone (3.34 g, 27 mmol) and N,N-dimethylbarbituric acid (4.2 g, 27 mmol) were added, followed by 15 mL of acetic anhydride. The reaction was stirred at 120 °C under argon protection for 5 h. After cooling to room temperature, the precipitate was filtered and washed with ethanol to remove acetic anhydride, obtaining 5.9 g of yellow powder solid, yield 83%, melting point: 265-267 °C.

[0043]

[0044] To a 100 mL round bottom flask, 2,6-dimethylpyranone (3.34 g, 27 mmol) and N,N-dimethylbarbituric acid (4.2 g, 27 mmol) were added, followed by 15 mL of acetic anhydride. The reaction was stirred at 120 °C under argon protection for 5 h. After cooling to room temperature, the precipitate was filtered and washed with ethanol to remove acetic anhydride, obtaining 5.9 g of yellow powder solid, yield 83%, melting point: 265-267 °C. + ] = 541.1876; Calcd for [M] = 541.1849. 1 H NMR (400 MHz, CDC13), δ (ppm): 9.19 (s, 2H), 9.10 (s, 2H), 8.36 (s, 2H), 8.15 (d, J = 8.4, 2H), 7.91 (d, J = 7.6, 2H), 7.80 (d, J = 7.6, 2H), 7.75 (d, J = 16.4, 2H), 7.64 (t, J = 7.2, 2H), 7.19 (d, J = 16.4, 2H), 3.40 (s, 6H); 13 C NMR (100 MHz, CDC13), δ (ppm): 164.27, 159.50, 149.40, 135.19, 133.94, 131.14, 129.82, 128.69, 128.24, 128.10, 128.04, 122.34, 113.32, 97.66, 28.56.

[0045]

[0046] To a 100 mL round bottom flask, 2,6-dimethylpyranone (3.34 g, 27 mmol) and N,N-dimethylbarbituric acid (4.2 g, 27 mmol) were added, followed by 15 mL of acetic anhydride. The reaction was stirred at 120 °C under argon protection for 5 h. After cooling to room temperature, the precipitate was filtered and washed with ethanol to remove acetic anhydride, obtaining 5.9 g of yellow powder solid, yield 83%, melting point: 265-267 °C. + ] = 541.1876; Calcd for [M] = 541.1849. 1H NMR (400MHz, DMSO-d6), δ (ppm): 10.15 (s, 2H), 9.48 (s, 2H), 8.83 (s, 2H), 8.31 (d, J = 9.8Hz, 2H), 8.08 (t, J = 7.2Hz, 2H), 8.01(d,J=7.6Hz,2H),7.89(d,J=16.4Hz,2H),7.83(d,J=7.2Hz,2H),7.69(d,J=16.0Hz,2H),4.61(s,6H),3.1(s,6H); 13 C NMR (100MHz, DMSO-d6), δ (ppm): 163.0, 158.3, 153.4, 150.0, 144.2, 136.2, 129.6, 129.0, 125.8, 119.4, 119.4, 113.9, 46.0, 28.2.

[0047] Example 2: Synthesis of probe TBA-γ-Q

[0048]

[0049] 0.6 g (5 mmol) of 2,6-dimethylpyranone and 1 g (5 mmol) of N,N-diethyl-thiobarbituric acid were added to a 100 mL round-bottom flask, followed by 15 mL of acetic anhydride. The mixture was stirred and refluxed at 120 °C for 5 h under argon protection. After cooling to room temperature, the precipitate was filtered and washed with ethanol to remove the acetic anhydride, yielding 0.98 g of a yellow powdery solid (65% yield) with a melting point of 195–197 °C.

[0050]

[0051] In a 50 mL round-bottom flask, compound A2 (200 mg, 1 mmol) and 3-quinoline carbaldehyde (90 mg, 0.91 mmol) were added, followed by 10 mL of ethanol and 20 μL of piperidine. The mixture was sealed and reacted at 90 °C for 48 h. The solvent was evaporated, and the solid was sonicated with acetonitrile and filtered to give 50 mg of a red solid (TBA-γ-q), yield: 30%. ESI: [M+H] + =585.1960; Calcd for [M+H] + :585.1956; 1H NMR (400 MHz, CDC13), δ (ppm): 9.22 (s, 2H), 9.12 (s, 2H), 8.39 (s, 2H), 8.17 (d, J = 7.2 Hz, 2H), 7.92 (d, J = 4.0 Hz, 2H), 7.81 (m, 4H), 7.65 (t, J = 5.6 Hz, 2H), 7.21 (d, J = 17.2 Hz, 2H), 4.62 (q, J = 4.8 Hz, 4H), 1.35 (s, 6H); 13 C NMR (100 MHz, DMSO-d6), δ (ppm): 161.8, 159.6, 149.0, 135.0, 134.1, 130.9, 128.4, 127.8, 127.8, 121.9, 113.8, 43.5, 12.6.

[0052]

[0053] In the Schlenk tube, 0.31 g of compound TBA-γ-q, 0.5 mL of iodomethane were added, sealed, protected from light, and reacted at 60 °C for 24 h. After stopping the reaction, 0.2 mL of fluoboric acid was added, stirred at room temperature for 2 h, and the solid was suction filtered. The obtained solid was again soaked in acetonitrile, ultrasonicated, suction filtered, and 0.04 mg of orange-red solid (TBA-γ-Q) was obtained, with a yield of 60%. 1 H NMR (400 MHz, DMSO-d6), δ (ppm): 10.163 (s, 2H), 9.591 (s, 2H), 8.950 (s, 2H), 8.38 (d, J = 8.4 Hz, 2H), 8.16 (t, J = 6.8 Hz, 4H), 8.08 (d, J = 16.8 Hz, 2H), 7.93 (t, J = 6 Hz, 2H), 7.82 (d, J = 16 Hz, 2H), 4.65 (s, 6H), 4.39 (q, J = 6.4 Hz, 4H), 1.20 (t, J = 6.4 Hz, 6H); 13 C NMR (100 MHz, DMSO-d6), δ (ppm): 177.3, 161.0, 144.5, 137.8, 136.3, 131.0, 129.6, 129.1, 125.6, 119.6, 114.9, 46.1, 43.3, 12.8.

[0054] Example 3: Viscosity dependence experiment of probe BBA-γ-Q

[0055] Glycerol-water solutions of 0%, 10%, 20%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, and 100% (0cp-1412cp) were prepared using glycerol and ultrapure water at different mass ratios (m / m). Equal volumes of the standard parent solution were added to 3 mL of each solution in systems of different viscosities to prepare 5 μM probe solutions. After shaking and mixing, their fluorescence emission spectra were measured. Figure 1 It can be observed that the probe BBA-γ-Q exhibits weak fluorescence in low-viscosity systems. This is because the rotation of the C-C single bond causes the probe to release intramolecular energy in a non-radiative manner, thus resulting in weak fluorescence intensity and low fluorescence quantum efficiency. As the system viscosity increases, the fluorescence intensity at 582 nm wavelength also increases continuously. In the high-viscosity glycerol system, the fluorescence intensity is 81 times that in pure water. This is because the rotation of the C-C single bond in the probe molecule is suppressed in high-viscosity systems, and the intramolecular excited-state energy is converted into fluorescence. These results indicate that the probe BBA-γ-Q is highly sensitive to viscosity within a certain range and can be used as a fluorescent probe for quantitative detection of viscosity changes in microenvironments.

[0056] Example 4: Continuous Fluorescent Titration of SO3 Using BBA-γ-Q Probe 2- experiment

[0057] from Figure 2 It can be observed that without the addition of SO3 2- At that time, it could be observed that the probe BBA-γ-Q itself exhibited weak fluorescence at 761 nm, and with SO3 2- As the concentration gradually increased, the fluorescence intensity of the probe at 761 nm gradually increased. This was further observed when 23 equivalents of SO3 were added. 2- When the fluorescence intensity tends to stabilize, the probe reacts with SO3. 2- The fitting constant of the post-reaction curve is greater than 0.99. The principle is that SO3... 2- It can attack the 4-position of the probe quinoline iodonium salt, undergoing a 1,4-Michael addition reaction, transforming the electron-withdrawing group of the quinoline iodonium salt into an electron-donating group, producing a significant fluorescent signal. Figure 3 It is known that the wavelength at which the probe BBA-γ-Q recognizes viscosity is 582 nm and the wavelength at which it interacts with sulfite is 761 nm. The wavelength difference between the two is 179 nm, which has a small wavelength overlap, thus providing detection sensitivity for practical applications and bioimaging detection.

[0058] Example 5: Probe BBA-γ-Q on SO3 2- Test strip experiment

[0059] BBA-γ-Q was made into a bisulfite test paper for monitoring the content of bisulfite in water. The test paper was prepared by soaking filter paper of the same size in a DMSO solution of BBA-γ-Q (5 mM) for 2 hours and then drying. A series of different contents of bisulfite in aqueous solution were added dropwise to the test paper strip, and a change from orange to purple was observed under visible light. The results show that BBA-γ-Q can be used as a tool for qualitative detection of SO2 derivatives by the naked eye in the environment (see Figure 4 ).

[0060] Example 6: Practical application of probe BBA-γ-Q

[0061] Various thickening agents are usually added to liquid foods to enhance the texture, consistency and uniformity of the liquid. Seven representative food thickeners (sodium carboxymethyl cellulose, pectin, gelatin, gum arabic, xanthan gum, sodium alginate and guar gum) were selected to study the applicability of probe BBA-γ-Q to their thickening effect. Different amounts (1.0-10.0 g / kg) of the seven food thickeners were prepared into various viscous solutions. The fluorescence intensity gradually increased with the amount of thickener added. As shown in Figure 5 , the mass concentration of the food thickener was linearly related to the fluorescence intensity. Xanthan gum had the highest thickening efficiency and sodium carboxymethyl cellulose had the lowest, indicating that the probe can be used as a molecular tool for fluorescence determination of the viscosity change caused by thickening agents.

[0062] Although the above disclosure is disclosed with preferred embodiments, it is not intended to limit the claims, and any person skilled in the art can make several possible changes and modifications without departing from the concept of the present application, therefore the protection scope of the present application should be subject to the scope defined by the claims of the present application.

Claims

1. A class of viscosity and SO2 dual-responsive fluorescent probes, characterized in that, The structure of the fluorescent probe is shown as formula (I): In formula (I), R is one of methyl or ethyl; X is one of S or O.

2. The preparation method of the viscosity and SO2 dual-responsive fluorescent probe according to claim 1, and the preparation general flowchart is shown as follows: The specific preparation steps are as follows: S1, 2, 6-dimethylpyranone and barbituric acid or derivatives are added into a round-bottom flask containing acetic anhydride, heated to reflux under the protection of nitrogen or argon, cooled, filtered, and yellow powdery solid is obtained, which is recrystallized with ethanol to obtain yellow crystalline powder A; S2, compound A and 3-quinoline formaldehyde are added into a round-bottom flask, ethanol is used as a reaction solvent and piperidine is used as an acid binding agent, the reaction is sealed and oscillated, after the reaction is stopped, the solid is extracted by filtration, and then the solid is immersed in acetonitrile and ultrasonically purified to obtain orange solid B; S3, compound B and iodomethane are added into a Schlenk tube, sealed and reacted in the dark, after the reaction is completed, fluoroboric acid is added, and after stirring at room temperature for 2 hours, the obtained solid is extracted by filtration, immersed in acetonitrile and ether, and ultrasonically purified to obtain red solid, which is the target probe molecule shown in formula (I).

3. The preparation method according to claim 2, characterized in that, In S1, the condensation reaction time is 5 hours and the temperature is 120°C; in S2, the condensation reaction time is 24-48 hours and the temperature is 90°C; in S3, the nitrogen methylation reaction time is 24 hours and the temperature is 60°C.

4. The preparation method according to claim 2, characterized in that, In S1, the feeding ratio of 2, 6-dimethylpyranone and barbituric acid or derivatives is 1:1-2; in S2, the condensation reaction, the feeding ratio of compound A and 3-quinoline formaldehyde is 1:2-2.5; the volume of piperidine added is 2‰; in S3, the feeding ratio of compound B and iodomethane in the nitrogen methylation reaction is 1:15-25.

5. The preparation method according to claim 2, characterized in that, In S1, compound A needs to be recrystallized with ethanol and extracted by filtration again; in S2, piperidine needs to be added as an acid binding agent to obtain compound B; in S3, fluoroboric acid needs to be added and stirred at room temperature to obtain the target compound. 6.The application of the class of viscosity and SO2 dual-responsive fluorescent probes according to claim 1, characterized in that, For the detection of SO2 and viscosity in food, water sample and food thickening agent, or made into test paper to detect the concentration of SO3 in liquid. 2- For the detection of SO2 and viscosity in food, water sample and food thickening agent, or made into test paper to detect the concentration of SO3 in liquid.

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