Pyrenyl Schiff base fluorescent derivative, fluorescent probe, preparation method of pyrenyl Schiff base fluorescent derivative and application of pyrenyl Schiff base fluorescent probe in biological

Abstract

The invention discloses a pyrenyl Schiff base fluorescent derivative, a fluorescent probe, a preparation method of the pyrenyl Schiff base fluorescent derivative and application of the pyrenyl Schiff base fluorescent probe in biological imaging, and belongs to the technical field of fluorescent probes. The pyrenyl Schiff base fluorescent probe capable of specifically detecting the zinc ions is prepared through two-step chemical reaction of Suzuki coupling reaction and aldehyde amine condensation reaction, and the pyrenyl Schiff base fluorescent probe has good recoverability and low cytotoxicity, is high in practicability and low in cost, and can be applied to detection of the zinc ions. In addition, the sensor has excellent ion selectivity to zinc ions, extremely high sensitivity, good Stokes displacement and visual sensing performance, and detection of exogenous zinc ions and endogenous zinc ions in cells and tissues is realized through biological imaging. The problems of difficult synthesis process, slow response speed, high detection cost, non-ideal biological imaging effect and the like of the conventional fluorescent derivative are solved.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent probes, and in particular relates to a pyrene-Schiff base fluorescent derivative, a fluorescent probe, a preparation method thereof, and biological imaging applications. Background Art

[0002] Zinc is the second most essential trace element after iron and is of vital importance in regulating various physiological reactions in the human body. Zinc homeostasis imbalance will affect the health of the organism. Studies have shown that zinc deficiency can lead to various clinical diseases such as reproductive dysfunction, immune system dysfunction, growth retardation and endocrine disorders; zinc excess can cause symptoms such as diarrhea, nausea, poisoning, and can also cause diseases such as coronary heart disease and arteriosclerosis; in addition, neurological diseases such as Parkinson's disease and Alzheimer's disease are also caused by zinc imbalance. Therefore, zinc has a great impact on the health of organisms. Effectively monitoring the concentration of zinc ions in organisms has important application value for the diagnosis, treatment and prevention of related diseases.

[0003] Detection of Zn 2+ The traditional techniques mainly include atomic absorption spectroscopy, spectrophotometry, chromatography, electrochemical analysis, inductively coupled plasma mass spectrometry, etc. Although these methods can selectively detect Zn 2+ And it has high sensitivity, but there are still many problems, such as: high cost of instruments and equipment, complicated operation process, long detection time, etc., which cannot meet the needs of real-time in-situ online monitoring, thus limiting its application in many fields. However, compared with the above methods, fluorescent probes not only have the advantages of good selectivity, high sensitivity, and high spatial resolution of traditional detection technology, but also have the advantages of simple operation, miniaturization and easy to carry, fast response speed, and convenient biological imaging and observation, which can make up for the shortcomings of traditional detection technology methods. Based on Zn 2+ The excellent performance of fluorescent probes has good application prospects in chemical analysis, life sciences, clinical medicine, environmental sciences and other fields.

[0004] According to literature statistics and research, about Zn 2+ Although the synthesis of fluorescent probes has been reported, there are no domestic reagents on the market and it is difficult to purchase them. 2+ Fluorescent probes have certain limitations. They are expensive and have a short effective time. In addition, a large number of fluorescent probes have been reported to have unsatisfactory effects in biological imaging and detection, so they are subject to certain restrictions in practical applications. 2+ The content and distribution of Zn is crucial. Therefore, it is necessary to design and synthesize Zn with the characteristics of simple synthesis, strong ion selectivity, good sensitivity, rapid response, stable photochemical properties, strong biocompatibility, and low cytotoxicity. 2+Fluorescent probes and their application in the biomedical field are of vital importance. Summary of the invention

[0005] In order to overcome the shortcomings of the above-mentioned prior art, the purpose of the present invention is to provide a pyrene Schiff base fluorescent derivative, a fluorescent probe and a preparation method and bio-imaging application thereof, aiming to solve the problems of poor selectivity, high detection cost, unsatisfactory bio-imaging effect and other problems existing in the existing detection methods that are difficult to meet the requirements of zinc ion recognition and monitoring.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect of the present invention, a pyrene-Schiff base fluorescent derivative is provided, wherein the general structural formula of the pyrene-Schiff base fluorescent derivative is as follows:

[0008]

[0009] Wherein, R is a substituted or unsubstituted o-hydroxy aromatic aldehyde, and the substituted or unsubstituted o-hydroxy aromatic aldehyde is one of the following structural formulas:

[0010]

[0011] In the above structural formula: R1, R2, R3 and R4 can be independently selected from hydrogen, hydroxyl, alkyl, alkoxy, halogen, substituted or unsubstituted C6~C 60 Aromatic ring group, substituted or unsubstituted C3~C 60 One of the aromatic heterocyclic groups.

[0012] The second aspect of the present invention provides a method for preparing a pyrene-Schiff base fluorescent derivative, comprising the following steps:

[0013] 1) In a nitrogen atmosphere, add 1-bromopyrene, 2-aminopyridine-5-boric acid pinacol ester, potassium carbonate aqueous solution, tetrakis(triphenylphosphine)palladium and 1,4-dioxane to a container equipped with a magnetic stirring and condensation reflux device, heat to 100-110°C (such as 100°C), stir and reflux for 24-36 hours (such as 24 hours) until the reaction is complete, slowly pour the reaction solution into an aqueous solution containing saturated sodium chloride, stir well, let stand, filter, wash with water and dry the filter cake. Use an eluent to separate and purify by column chromatography, and remove the organic solvent by rotary evaporation to obtain the intermediate Py-NH2.

[0014] 2) In a nitrogen atmosphere, the intermediate Py-NH2, the raw material X, glacial acetic acid and anhydrous ethanol are added to a container equipped with a magnetic stirring and condensation reflux device, and the temperature is raised to 78-85° C. (e.g., 80° C.), and the mixture is stirred and refluxed for 2-4 hours (e.g., 3 hours). After 3 hours, a large amount of solid is precipitated in the reaction solution, which is filtered while hot and washed with anhydrous ethanol to obtain a crude target product, which is heated and dissolved with anhydrous ethanol, filtered while hot, and further purified to obtain the target product, a pyrenyl Schiff base fluorescent derivative.

[0015] Wherein, the structural formula of the raw material X is R=O, R is a substituted or unsubstituted o-hydroxy aromatic aldehyde, and the substituted or unsubstituted o-hydroxy aromatic aldehyde is one of the following structural formulas:

[0016]

[0017] In the above structural formula: R1, R2, R3 and R4 can be independently selected from hydrogen, hydroxyl, alkyl, alkoxy, halogen, substituted or unsubstituted C6~C 60 Aromatic ring group, substituted or unsubstituted C3~C 60 One of the aromatic heterocyclic groups.

[0018] Furthermore, in step (1), the molar ratio of 1-bromopyrene, 2-aminopyridine-5-boric acid pinacol ester, potassium carbonate aqueous solution, tetrakis(triphenylphosphine)palladium and 1,4-dioxane is 1:(1.1-1.3):(3-5):(0.05-0.15):(80-120).

[0019] Furthermore, the eluent for column chromatography separation is a dichloromethane-methanol system with a volume ratio of (90-110):1.

[0020] Furthermore, the volume ratio of the eluent dichloromethane to methanol is 100:1.

[0021] Furthermore, in step (2), the molar ratio of the intermediate Py-NH2, the raw material X and anhydrous ethanol is 1:(1.1-1.3):(20-30).

[0022] Furthermore, the molar ratio of the intermediate Py-NH2, the raw material X and anhydrous ethanol is 1:1.1:20.

[0023] The third aspect of the present invention provides a method for preparing a fluorescent probe from a pyrene Schiff base fluorescent derivative, which comprises the following steps:

[0024] (1) The above-mentioned pyrene Schiff base fluorescent derivative is dissolved in an organic solvent (such as dimethyl sulfoxide, N,N-dimethylformamide, etc.) to prepare 1×10 -2 mol / L~5×10 -2mol / L (e.g. 1×10 -2 mol / L) fluorescent probe mother solution for standby use;

[0025] (2) Add 2997 μL of an organic solvent (such as dimethyl sulfoxide, N,N-dimethylformamide, etc.), ultrapure water, or one of the buffer solutions (such as PBS buffer solution, Tris-HCl buffer solution) to 3 μL of the fluorescent probe mother solution to dilute the fluorescent probe mother solution to 1×10 -5 mol / L~5×10 -5 mol / L (e.g. 1×10 -5 mol / L) solution, shake it well, and place it in a dark environment at room temperature for 2 hours to prepare a fluorescent probe.

[0026] In a fourth aspect of the present invention, a fluorescent probe is prepared by the above preparation method.

[0027] Dissolve zinc acetate in ultrapure water to prepare a 1 mol / L zinc acetate mother solution, and then dilute the zinc acetate mother solution to 1×10 -3 ~5×10 -3 mol / L zinc acetate solution;

[0028] Different metal interfering ions (Mn 2+ , Na + , Cd 2+ , K + , Pb 2+ , Ba 2+ , Li + ,Mg 2+ , Al 3+ , Ca 2+ , Ag + , Hg 2+ , Sr. 2+ , Cu + Dissolve the metal ions in ultrapure water to prepare a 1 mol / L ion stock solution, and then dilute the ion stock solution to 5×10 -3 mol / L metal interfering ion solution.

[0029] The fluorescent probe prepared in the third aspect of the present invention was accurately pipetted into 3 mL of 1×10 -5 mol / L~5×10 -5 mol / L (e.g. 1×10 -5 mol / L) in a quartz cuvette, and then add 3 μL of 1×10 -3 ~5×10 -3 mol / L (e.g. 1×10 -5mol / L) zinc acetate solution, and tested its UV-visible titration and fluorescence titration spectra respectively until its UV or fluorescence spectrum no longer changed, in order to explore the optical properties of the fluorescent probe for zinc ion sensing.

[0030] The fluorescent probe prepared in the third aspect of the present invention was accurately pipetted into 3 mL of 1×10 -5 mol / L~5×10 -5 mol / L (e.g. 1×10 -5 mol / L) in a quartz cuvette, and 3eq. of the above-mentioned different types of metal interfering ion solutions were added respectively, and their fluorescence emission spectra were tested; then 3eq. of zinc acetate solution was added thereto, and its fluorescence emission spectrum was tested to explore the selectivity and anti-interference ability of the fluorescent probe for zinc ions.

[0031] The present invention uses the above fluorescent probe solution and ion solution to conduct experiments, and the experimental results show that Zn 2+ After addition, the fluorescence intensity of the fluorescent probe solution increases rapidly. It is a typical "Turn-on" type fluorescent probe with good selectivity and ion anti-interference for zinc ions.

[0032] A fifth aspect of the present invention provides an application of the fluorescent probe as described above for biological imaging of exogenous / endogenous zinc ions.

[0033] The object to be treated is incubated in the dark with the fluorescent probe prepared in the third aspect of the present invention for 30 to 50 minutes (eg 35 minutes), and live cell imaging is performed using a laser confocal fluorescence microscope.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] The present invention discloses a pyrene-based Schiff base fluorescent derivative. The structural design of the derivative enables it to be used as a fluorescent probe. In particular, by introducing ortho-hydroxy aromatic aldehydes with different molecular structures, its optical properties can be adjusted, providing a basis for subsequent fluorescence detection. In addition, the derivative can react with Zn through the combined action of the nitrogen atom in pyridine, the nitrogen atom in the Schiff base and the oxygen atom in the ortho-hydroxyl group. 2+ Specific chelation forms a coordination molecule; at the same time, the fluorescent group pyrene and other groups produce conjugation, thereby changing the basic photophysical properties of the fluorescent probe and reacting with Zn 2+ After combination, it can produce a specific fluorescence phenomenon, thus achieving the purpose of Zn 2+ This property makes the derivative a suitable candidate for detecting Zn 2+ A potentially powerful tool with high selectivity and sensitivity.

[0036] The invention discloses a method for preparing a pyrene-based Schiff base fluorescent derivative. The Schiff base fluorescent derivative containing an imine group is synthesized by Suzuki coupling reaction and aldehyde-amine condensation reaction. 2+The formation of coordination molecules produces a specific fluorescence phenomenon. First, 1-bromopyrene is selected as a halogenated hydrocarbon, 2-aminopyridine-5-boronic acid pinacol ester is used as an aromatic borate ester, and tetrakis(triphenylphosphine)palladium is used as a transition metal catalyst to prepare the intermediate Py-NH2 through a Suzuki coupling reaction. Among them: (1) The bromine atom in the molecular structure of 1-bromopyrene is the key site of the Suzuki coupling reaction. The presence of the bromine atom enables 1-bromopyrene to undergo a cross-coupling reaction with aromatic borate esters (such as 2-aminopyridine-5-boronic acid pinacol ester) in the presence of the transition metal catalyst tetrakis(triphenylphosphine)palladium to form a CC bond, thereby constructing the skeleton structure of the target product; (2) In addition to participating in the coupling reaction, 2-aminopyridine-5-boronic acid pinacol ester also introduces an aminopyridine group into the target product, which has an important influence on the fluorescence properties and biological activity of the product. (3) The addition of potassium carbonate aqueous solution provides the necessary alkaline environment for the reaction. In the Suzuki coupling reaction, alkaline conditions, on the one hand, help promote the deprotonation of aromatic borate esters to form borate ions that are more reactive, thereby accelerating the coupling reaction; on the other hand, they can also promote the activity of the catalyst, reduce the activation energy of the reaction, allow the reaction to proceed under milder conditions, and improve the reaction rate and yield. (4) Tetrakis(triphenylphosphine)palladium is the key catalyst in the preparation method. On the one hand, it can form an active catalytic center in the reaction system to promote the cross-coupling reaction between 1-bromopyrene and 2-aminopyridine-5-boronic acid pinacol ester. On the other hand, it can accurately identify and catalyze the target reaction in a complex reaction system, while inhibiting the occurrence of side reactions, thereby improving the selectivity of the reaction and the purity of the product. (5) 1,4-dioxane is used as a solvent, and the chemical properties of 1,4-dioxane are relatively stable. During the preparation process, it can not only maintain the stability of the reaction system, provide a suitable reaction environment, and avoid unnecessary side reactions, but also promote contact and collision between reactants, accelerate the reaction, and thus facilitate the formation of the target product. The intermediate Py-NH2 also plays an important role in the preparation method: on the one hand, the amino functional group in Py-NH2 has strong reactivity and can participate in the condensation reaction. On the other hand, the presence of the pyridine ring makes the Py-NH2 compound have good thermal stability and chemical stability. Secondly, the intermediate Py-NH2, raw material X, glacial acetic acid and anhydrous ethanol are mixed, and the target product, the pyrenyl Schiff base fluorescent derivative, is obtained by heating, filtering and other treatments. In this step, the amino group in the intermediate Py-NH2 undergoes a condensation reaction with the aldehyde group in the raw material X to generate a Schiff base structure containing an imine group (-C=N-), i.e., the target product. Experiments have confirmed that the pyrenyl Schiff base fluorescent derivative prepared above has good photochemical stability and successfully avoids photobleaching, and has the advantages of good ion selectivity and strong ion anti-interference ability.The pyrene-Schiff base fluorescent derivative prepared by the present invention can specifically identify and detect zinc ions, and exhibits excellent properties such as fast response speed, low detection limit, good recoverability and large Stokes shift, etc., solving the problems of poor selectivity, high detection cost and unsatisfactory biological imaging effect existing in the existing detection methods.

[0037] Furthermore, the present invention prepares a pyrene-based Schiff base fluorescent probe that can specifically detect zinc ions through two-step chemical reactions of Suzuki coupling reaction and aldehyde-amine condensation reaction, which can achieve rapid and visual detection of zinc ions and has good recoverability and low cytotoxicity, high practicality, low cost, huge advantages and potential application value in the biomedical field. The fluorescent probe prepared by the present invention using metal ion chelation coordination and fluorescence turn-on effect has excellent photochemical stability, extremely high sensitivity and good ion selectivity. The series of fluorescent probes combined with fluorescence instruments can achieve efficient and rapid identification and detection of zinc ions in solutions and organisms.

[0038] Furthermore, the present invention uses the above-mentioned pyrene-Schiff base fluorescent derivatives to perform bioimaging applications on endogenous zinc ions and exogenous zinc ions in living cells and tissues. Experimental results show that the above-mentioned pyrene-Schiff base fluorescent derivatives can perform visual detection of endogenous zinc ions and exogenous zinc ions in living cells and tissues in biological systems, which solves the shortcomings of existing bioimaging effects that are not ideal, and provides new research ideas for the design, synthesis and bioimaging applications of zinc ion fluorescent probes. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 is the H NMR spectrum of the fluorescent compound Py-A obtained in Example 6 of the present invention;

[0040] Figure 2 is the NMR carbon spectrum of the fluorescent compound Py-A obtained in Example 6 of the present invention;

[0041] Figure 3 is a high-resolution mass spectrum of the fluorescent compound Py-A obtained in Example 6 of the present invention;

[0042] Figure 4 The UV-visible absorption and fluorescence emission spectra of the fluorescent compound Py-A obtained in Example 6 of the present invention;

[0043] Figure 5 is a photochemical stability spectrum of the fluorescent compound Py-A obtained in Example 6 of the present invention in DMSO;

[0044] Figure 6The UV-visible titration and fluorescence titration spectra of the fluorescent probe Py-A obtained in Example 6 of the present invention sensing zinc ions in DMSO;

[0045] Figure 7 The selectivity and anti-interference spectrum of the fluorescent probe Py-A obtained in Example 6 of the present invention for metal ion sensing in DMSO;

[0046] Figure 8 This is an optical photograph of the selectivity and anti-interference of the fluorescent probe Py-A obtained in Example 6 of the present invention for metal ion sensing in DMSO;

[0047] Fig. 9 This is a repeatability spectrum of the fluorescent probe Py-A obtained in Example 6 of the present invention in response to zinc ions;

[0048] Fig.10 The recognition mechanism of the fluorescent probe Py-A obtained in Example 6 of the present invention in response to zinc ions;

[0049] Fig.11 This is a fluorescence imaging diagram of the fluorescent probe Py-A obtained in Example 6 of the present invention detecting exogenous zinc ions in an organism (HeLa cells);

[0050] Fig.12 This is a fluorescence imaging diagram of the fluorescent probe Py-A obtained in Example 6 of the present invention detecting exogenous zinc ions in an organism (KGN cells);

[0051] Fig.13 This is a fluorescence imaging diagram of the fluorescent probe Py-A obtained in Example 6 of the present invention for detecting endogenous zinc ions in an organism. DETAILED DESCRIPTION

[0052] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0053] It should be noted that the terms "first", "second", etc. in the specification and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units that are clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0054] In order to better illustrate the present invention, specific examples will be used to further illustrate the present invention.

[0055] Example 1

[0056] This embodiment provides an intermediate compound Py-NH2, whose structural formula is shown below:

[0057]

[0058] The preparation method of the intermediate compound Py-NH2 is as follows:

[0059] Add 2.81g of 1-bromopyrene, 2.42g of 2-aminopyridine-5-boric acid pinacol ester, 10mL of 5M potassium carbonate aqueous solution, 25mg of tetrakis(triphenylphosphine)palladium and 100mL of 1,4-dioxane as reaction solvent to a 250mL two-necked round-bottom flask, stir and reflux under nitrogen atmosphere at 100°C, and monitor by thin layer chromatography (TCL). Stop the reaction after 24 hours, slowly pour the reaction solution into an aqueous solution containing saturated sodium chloride, stir well and let stand, filter, wash and dry the filter cake. Use dichloromethane-methanol volume ratio of 100:1 as eluent, separate and purify with 200-300 mesh silica gel by column chromatography, remove the organic solvent by rotary evaporation, and obtain pale yellow solid Py-NH2 with a yield of 77%.

[0060] 1 H NMR (400MHz, DMSO-d6): δ8.33(d,1H),8.31(d,1H),8.28(d,1H),8.20(d,2H),8.19(d, 1H),8.17(s,2H),8.08(t,1H),7.99(d,1H),7.69(d,1H),6.70(d,1H),6.22(s,2H)ppm. 13C NMR (101MHz, DMSO-d6): δ159.6,149.2,139.4,135.4,131.5,131.0,130.2,128.3,128.1,128.0,127.9,127.6,126.9,125 .7,125.5,125.3,125.1,124.8,124.6,124.4,108.2,40.6,40.4,40.2,40.0,39.8,39.6,39.4ppm.ESI-HRMS(m / z):[M+H] + calcd:295.1230,found:295.1230.

[0061]

[0062] Example 2

[0063] This embodiment provides a compound, the structural formula of the compound is the same as that of the compound in Example 1. In the preparation method of the compound, the molar ratio of 1-bromopyrene, 2-aminopyridine-5-boric acid pinacol ester, potassium carbonate aqueous solution, tetrakis(triphenylphosphine)palladium and 1,4-dioxane is 1:1.1:3:0.05:80, and the remaining steps are consistent with Example 1.

[0064] Example 3

[0065] This embodiment provides a compound, the structural formula of the compound is the same as that of the compound in Example 1. In the preparation method of the compound, the molar ratio of 1-bromopyrene, 2-aminopyridine-5-boric acid pinacol ester, potassium carbonate aqueous solution, tetrakis(triphenylphosphine)palladium and 1,4-dioxane is 1:1.3:5:0.15:120, and the remaining steps are consistent with Example 1.

[0066] Example 4

[0067] This embodiment provides a compound, the structural formula of the compound is the same as that of the compound in Embodiment 1. In the preparation method of the compound, except that the volume ratio of the eluent dichloromethane-methanol for column chromatography separation is 90:1, the remaining steps are consistent with those in Embodiment 1.

[0068] Example 5

[0069] This embodiment provides a compound, the structural formula of the compound is the same as that of the compound in Embodiment 1. In the preparation method of the compound, except that the volume ratio of dichloromethane-methanol for column chromatography separation is 110:1, the remaining steps are consistent with those in Embodiment 1.

[0070] By comparing the preparation methods of compound Py-NH2 in Examples 1-5, the results are basically consistent, so the volume ratio of dichloromethane to methanol for column chromatography separation is selected as an integer of 100:1.

[0071] Example 6

[0072] This embodiment provides a compound Py-A, whose structural formula is shown below:

[0073]

[0074] The preparation method of compound Py-A is as follows:

[0075] Add 0.29g of Py-NH2 and 0.13g of raw material A, 1 drop of glacial acetic acid, and 20mL of anhydrous ethanol as the reaction solvent to a 50mL two-necked round-bottom flask, and stir and reflux under nitrogen atmosphere at 80°C. At the beginning of the reaction, the solution is yellow in color, and the solid can be completely dissolved after heating for 5-10min. It is monitored by thin layer chromatography (TCL). After 3 hours, the reaction is complete, and a large amount of solid precipitates in the reaction solution. Filter while hot, and wash with anhydrous ethanol for more than 3 times to obtain an orange-yellow solid.

[0076] The obtained solid was recrystallized from anhydrous ethanol to obtain further purified orange-yellow solid Py-A with a yield of 83%.

[0077] 1 H NMR (400MHz, DMSO-d6): δ13.08(s,1H),9.67(s,1H),8.82(s,1H),8.44(d,1H),8.38(d,1H),8.35(d,1H),8. 28(s,2H),8.27-8.22(m,2H),8.16-8.11(m,3H),7.87(dd,1H),7.73(d,1H),7.51(t,1H),7.04(dd,2H)ppm. 13 C NMR (101MHz, CDCl3): δ164.8,162.0,156.6,150.2,140.3,136.0,134.0,133.6,133.1,131.5,131.3,130.9,128.8,128.3,128.0,127 .6,127.4,126.3,125.6,125.3,125.1,124.9,124.8,124.4,120.2,119.4,119.2,117.4,77.4,77.1,76.8ppm.ESI-HRMS(m / z):[M+H] + calcd:399.1492,found:399.1490.

[0078]

[0079] Example 7

[0080] This embodiment provides a compound, the structural formula of the compound is the same as that of the compound in Example 6. In the preparation method of the compound, the molar ratio of Py-NH2, raw material A and anhydrous ethanol is 1:1.1:20, and the remaining steps are consistent with Example 6.

[0081] Example 8

[0082] This embodiment provides a compound, the structural formula of the compound is the same as that of the compound in Example 6. In the preparation method of the compound, the molar ratio of Py-NH2, raw material A and anhydrous ethanol is 1:1.2:25, and the remaining steps are consistent with Example 6.

[0083] Example 9

[0084] This embodiment provides a compound, the structural formula of the compound is the same as that of the compound in Example 6. In the preparation method of the compound, the molar ratio of Py-NH2, raw material A and anhydrous ethanol is 1:1.3:30, and the other steps are consistent with Example 6.

[0085] By comparing the preparation methods of compound Py-A in Examples 6-9, the results produced by the preparation method of Example 6 are basically consistent with those of the preparation methods of Examples 7-9, so a molar ratio of 1:1.1:20 is selected to save more raw materials and reagents.

[0086] Example 10

[0087] This embodiment provides a compound Py-B, whose structural formula is shown below:

[0088]

[0089] The preparation method of compound Py-B is as follows:

[0090] Py-NH2 was prepared by the preparation method of the intermediate in Example 1;

[0091] In a 50mL two-necked round-bottom flask, add 0.29g of Py-NH2 and 0.19g of raw material B, 1 drop of glacial acetic acid, and 20mL of anhydrous ethanol as the reaction solvent, and stir and reflux under nitrogen atmosphere at 80°C. Monitored by thin layer chromatography (TCL), the reaction is complete after 3 hours, and a large amount of solid precipitates in the reaction liquid. Filter while hot, wash the filter cake 3 times with anhydrous ethanol to obtain an orange-yellow solid.

[0092] The obtained solid was recrystallized from anhydrous ethanol to obtain further purified orange-yellow solid Py-B with a yield of 78%.

[0093] 1 H NMR (400MHz, DMSO-d6): δ15.35(s,1H),10.01(s,1H),8.80(s,1H),8.44(d,1H),8.36(dd,3H),8.26(d ,4H),8.14(dd,3H),7.95(d,1H),7.90(d,1H),7.77(d,1H),7.58(t,1H),7.37(t,1H),6.92(d,1H)ppm. 13 C NMR (101MHz, CDCl3-d6): δ178.5,151.9,150.3,150.2,140.5,139.4,134. 5,134.1,133.0,131.5,131.3,130.9,129.4,128.8,128.6,128.3,128.0,1 27.6,127.4,127.1,126.3,125.6,125.3,125.1,124.9,124.8,124.7,124. 3,124.1,119.5,115.7,109.1,77.4,77.1,76.8ppm.ESI-HRMS(m / z):[M+H] + calcd:449.1648,found:449.1647.

[0094]

[0095] Embodiment 11

[0096] This embodiment provides a compound Py-C, whose structural formula is shown below:

[0097]

[0098] The preparation method of compound Py-C is as follows:

[0099] Py-NH2 was prepared by the preparation method of the intermediate in Example 1;

[0100] In a 50mL two-necked round-bottom flask, add 0.29g of Py-NH2 and 0.30g of raw material C, 1 drop of glacial acetic acid, and 20mL of anhydrous ethanol as the reaction solvent, and stir and reflux under nitrogen atmosphere at 80°C. Monitored by thin layer chromatography (TCL), the reaction is complete after 3 hours, and a large amount of solid precipitates in the reaction liquid. Filter while hot, wash the filter cake 3 times with anhydrous ethanol to obtain solid compound Py-C.

[0101] The obtained solid was recrystallized from anhydrous ethanol to obtain further purified compound Py-C with a yield of 82%.

[0102]

[0103] Example 12

[0104] This embodiment provides a compound Py-D, whose structural formula is shown below:

[0105]

[0106] The preparation method of compound Py-D is as follows:

[0107] Py-NH2 was prepared by the preparation method of the intermediate in Example 1;

[0108] In a 50mL two-necked round-bottom flask, add 0.29g of Py-NH2 and 0.27g of raw material D, 1 drop of glacial acetic acid, and 20mL of anhydrous ethanol as the reaction solvent, and stir and reflux under nitrogen atmosphere at 80°C. Monitored by thin layer chromatography (TCL), the reaction is complete after 3 hours, and a large amount of solid precipitates in the reaction liquid. Filter while hot, wash the filter cake 3 times with anhydrous ethanol to obtain solid compound Py-D.

[0109] The obtained solid was recrystallized from anhydrous ethanol to obtain further purified compound Py-D with a yield of 86%.

[0110]

[0111] Embodiment 13

[0112] This embodiment provides a compound Py-E, whose structural formula is shown below:

[0113]

[0114] The preparation method of compound Py-E is as follows:

[0115] Py-NH2 was prepared by the preparation method of the intermediate in Example 1;

[0116] In a 50mL two-necked round-bottom flask, add 0.29g of Py-NH2 and 0.32g of raw material E, 1 drop of glacial acetic acid, and 20mL of anhydrous ethanol as the reaction solvent, and stir and reflux under nitrogen atmosphere at 80°C. Monitored by thin layer chromatography (TCL), the reaction is complete after 3 hours, and a large amount of solid precipitates in the reaction liquid. Filter while hot, wash the filter cake 3 times with anhydrous ethanol to obtain solid compound Py-E.

[0117] The obtained solid was recrystallized from anhydrous ethanol to obtain further purified compound Py-E with a yield of 84%.

[0118]

[0119] Embodiment 14

[0120] This embodiment provides a compound Py-F, whose structural formula is shown below:

[0121]

[0122] The preparation method of compound Py-F is as follows:

[0123] Py-NH2 was prepared by the preparation method of the intermediate in Example 1;

[0124] In a 50mL two-necked round-bottom flask, add 0.29g of Py-NH2 and 0.26g of raw material F, 1 drop of glacial acetic acid, and 20mL of anhydrous ethanol as the reaction solvent, and stir and reflux under nitrogen atmosphere at 80°C. Monitored by thin layer chromatography (TCL), the reaction is complete after 3 hours, and a large amount of solid precipitates in the reaction liquid. Filter while hot, wash the filter cake 3 times with anhydrous ethanol to obtain solid compound Py-F.

[0125] The obtained solid was recrystallized from anhydrous ethanol to obtain further purified compound Py-F with a yield of 80%.

[0126]

[0127] Embodiment 15

[0128] This embodiment provides a compound Py-G, whose structural formula is shown below:

[0129]

[0130] The preparation method of compound Py-G is as follows:

[0131] Py-NH2 was prepared by the preparation method of the intermediate in Example 1;

[0132] In a 50mL two-necked round-bottom flask, add 0.29g of Py-NH2 and 0.19g of raw material G, 1 drop of glacial acetic acid, and 20mL of anhydrous ethanol as the reaction solvent, and stir and reflux under nitrogen atmosphere at 80°C. Monitored by thin layer chromatography (TCL), the reaction is complete after 3 hours, and a large amount of solid precipitates in the reaction liquid. Filter while hot, wash the filter cake 3 times with anhydrous ethanol, and obtain solid compound Py-G.

[0133] The obtained solid was recrystallized from anhydrous ethanol to obtain further purified compound Py-G with a yield of 82%.

[0134]

[0135] Embodiments 16 to 25

[0136] Examples 16 to 25 provide a fluorescent probe, respectively, using the compounds prepared in Examples 6 to 15 as raw materials, and the specific operation steps are as follows:

[0137] Step 1: Dissolve the compounds in dimethyl sulfoxide (DMSO) to prepare 1×10 -2 mol / L fluorescent probe stock solution, for later use;

[0138] Step 2: Add 2997 μL of DMSO to 3 μL of the fluorescent probe stock solution to dilute the fluorescent probe stock solution to 1×10 -5 mol / L solution, shake it well, and place it in a dark environment at room temperature for 2 hours to prepare a fluorescent probe.

[0139] Embodiments 26 to 35

[0140] Examples 26 to 35 provide a fluorescent probe, respectively, using the compounds prepared in Examples 6 to 15 as raw materials, and the specific operation steps are as follows:

[0141] Step 1: Dissolve the compounds in dimethyl sulfoxide (DMSO) to prepare 5×10 -2 mol / L fluorescent probe stock solution, for later use;

[0142] Step 2: Add 2997 μL of DMSO to 3 μL of the fluorescent probe stock solution to dilute the fluorescent probe stock solution to 5×10 -5 mol / L solution, shake it well, and place it in a dark environment at room temperature for 2 hours to prepare a fluorescent probe.

[0143] Embodiments 36 to 45

[0144] Examples 36 to 45 provide a fluorescent probe, respectively, using the compounds prepared in Examples 6 to 15 as raw materials, and the specific operation steps are as follows:

[0145] Step 1, the compounds were dissolved in N,N-dimethylformamide (DMF) to prepare 1×10 -2 mol / L fluorescent probe stock solution, for later use;

[0146] Step 2: Add 2997 μL of DMF to 3 μL of the fluorescent probe stock solution to dilute the fluorescent probe stock solution to 1×10 -5 mol / L solution, shake it well, and place it in a dark environment at room temperature for 2 hours to prepare a fluorescent probe.

[0147] Embodiments 46 to 55

[0148] Examples 46 to 55 provide a fluorescent probe, respectively, using the compounds prepared in Examples 6 to 15 as raw materials, and the specific operation steps are as follows:

[0149] Step 1, the compounds were dissolved in N,N-dimethylformamide (DMF) to prepare 5×10 -2 mol / L fluorescent probe stock solution, for later use;

[0150] Step 2: Add 2997 μL of DMF to 3 μL of the fluorescent probe stock solution to dilute the fluorescent probe stock solution to 5×10 -5 mol / L solution, shake it well, and place it in a dark environment at room temperature for 2 hours to prepare a fluorescent probe.

[0151] Embodiments 56 to 65

[0152] Examples 56 to 65 provide a fluorescent probe for biological imaging of HeLa cells and KGN cells, respectively, which are imaged using the fluorescent probes prepared in Examples 16 to 25, and the specific operation steps are as follows:

[0153] The fluorescent probes (1×10 -5 mol / L) were incubated in the dark for 35 min in HeLa cells and KGN cells, and live cell imaging was performed using a laser confocal fluorescence microscope. 2+ The aqueous solution was incubated in the dark for 35 min, and live cell imaging was performed again using a laser confocal fluorescence microscope.

[0154] Embodiments 66 to 75

[0155] Examples 66 to 75 provide a fluorescent probe for biological imaging of human sperm and rat testicular tissue frozen sections, respectively, which are imaged using the fluorescent probes prepared in Examples 16 to 25, and the specific operating steps are as follows:

[0156] The fluorescent probes (1×10 -5 mol / L) were incubated in the dark for 35 min, and biological imaging was performed using a laser confocal fluorescence microscope. Human sperm and rat testicular tissue that were not incubated with fluorescent probes for 35 min were also imaged using a laser confocal fluorescence microscope.

[0157] Test example:

[0158] In order to verify the technical effect of the present invention, a large number of systematic studies and applications were conducted on the detection of zinc ions by the pyrene Schiff base fluorescent derivative Py-A prepared in Example 6. The results of various experiments are shown below:

[0159] The structure of the fluorescent compound Py-A was strictly characterized by H-NMR, C-NMR and high-resolution mass spectrometry, such as Figure 1-3 .

[0160] Figure 4 The excitation emission spectrum of the fluorescent compound Py-A in dimethyl sulfoxide solution, where the solid line is the UV-visible absorption spectrum and the dotted line is the fluorescence emission spectrum. It can be seen from the figure that the maximum absorption wavelength of Py-A appears at 362nm and the maximum emission wavelength appears at 469nm.

[0161] Figure 5 The photochemical stability of the fluorescent compound Py-A in dimethyl sulfoxide solution was studied. The results showed that it has good photochemical stability and successfully prevented photobleaching, which laid a solid foundation for its practical application in sensing.

[0162] Figure 6 The UV-visible titration spectrum (a) and fluorescence titration spectrum (b) of the fluorescent compound Py-A sensing zinc ions in dimethyl sulfoxide solution. The UV-visible titration spectrum shows that in the presence of Zn 2+ After being gradually added to the Py-A solution, the absorption peak at 362nm gradually blue-shifted to 354nm, and the absorption intensity gradually decreased. A new absorption peak appeared at 446nm, and an isosbestic point appeared at 402nm. These phenomena indicate that the addition of Zn 2+ In addition, the Py-A solution itself is colorless when observed with the naked eye. 2+ After that, the solution gradually turned light yellow, indicating that Py-A 2+ The detection has a certain visual effect. The fluorescence titration spectrum shows that with the increase of Zn 2+ With the addition of 0-10 μM Zn, the fluorescence intensity of the Py-A solution gradually increased, and the emission wavelength gradually shifted from 469 nm to 521 nm, a 52 nm red shift occurred, and its fluorescence color quickly changed to bright green. 2+ Its fluorescence intensity showed a good linear relationship in the presence of Zn. Its detection limit was 29.15nM, which is much lower than the zinc content stipulated by the World Health Organization (WHO) and the zinc content in the human body, and is suitable for Zn 2+ Detection.

[0163] Figure 7 The ion selectivity (a) and ion interference resistance (b) of the fluorescent compound Py-A were studied by fluorescence spectroscopy. 2+ , Mn 2+ , Na + , Cd 2+ , K+ , Pb 2+ , Ba 2+ , Li + ,Mg 2+ , Al 3+ , Ca 2+ , Ag + , Hg 2+ , Sr. 2 + , Cu + When introduced into the Py-A solution, in addition to Zn 2+ The addition of other metal ions did not cause any changes in the fluorescence spectrum. 2+ When the Py-A solution was added, it turned yellow and produced bright green fluorescence. After the other metal ions were added, the color of the solution under sunlight and fluorescence remained basically unchanged. When an equal amount of zinc ions were added to the solution containing the above-mentioned metal interfering ions, the solution was not interfered by them and produced bright green fluorescence. The visualized optical picture is shown in Figure 8 The above results show that Py-A has a great influence on Zn 2+ It has good ion selection ability and anti-ion and anti-interference ability.

[0164] Fig. 9 The repeatability of the fluorescent compound Py-A in sensing zinc ions was investigated. When zinc ions were added to the Py-A solution, its fluorescence intensity increased rapidly. After adding ethylenediaminetetraacetic acid (EDTA), its fluorescence could basically be restored to its original state. After repeated addition of zinc ions and EDTA, the fluorescence intensity of the solution could still be restored to the fluorescence intensity of the first addition of zinc ions and the compound Py-A itself. This result shows that Py-A has good reusability, which is of necessary value and significance for the practical application of fluorescent probes.

[0165] Fig.10 The binding ratio of the fluorescent compound Py-A to zinc ions was explored through Job's curve and NMR titration spectrum. Job's curve (a) shows that the horizontal coordinate corresponding to the highest point of fluorescence intensity is 0.5; at the same time, the NMR titration spectrum (b) shows that after adding 1eq. of zinc ions, adding more zinc ions to it, the displacement of all hydrogens in the NMR spectrum no longer changes. The above experimental exploration shows that the compound binds to zinc ions in a 1:1 ratio.

[0166] Fig.11 and Fig.12 In order to explore the application of fluorescent probe Py-A in biological imaging by adding exogenous zinc ions, 50 μM Zn 2+After incubating Hela cells or KGN cells with 10μM Py-A or 10μM probe for 35min, no fluorescence signal was found after laser confocal fluorescence microscopy imaging; after incubating Hela cells or KGN cells with 10μM Py-A and 50μM zinc ions for 35min, bright green fluorescence was generated, and the bright field and dark field were perfectly overlapped after superposition, indicating that Py-A can be used to detect exogenous zinc ions in living cells.

[0167] Fig.13 The fluorescent probe Py-A was used to detect and image endogenous zinc ions in human sperm and rat testicular tissue. The frozen sections of human sperm and rat testicular tissue were incubated with 10 μM probe Py-A for 35 minutes and imaged on a laser confocal fluorescence microscope. The results are shown in the figure. Human sperm or rat testicular tissue incubated with Py-A can produce green fluorescence, while those not incubated with the fluorescent probe do not produce fluorescence. The results show that Py-A can be used to detect endogenous zinc ions in organisms.

[0168] In summary, the present invention designs and synthesizes a series of novel Schiff base fluorescent derivatives based on the fluorescent group pyrene. The synthesis process of this type of fluorescent derivative is simple and the yield is high. It has good photochemical stability and successfully avoids the photobleaching phenomenon. It also has the advantages of good ion selectivity and ion anti-interference ability. The fluorescent probe prepared based on this design has high sensitivity and high selectivity to zinc ions. It is a typical "Turn-on" type fluorescent probe suitable for the detection of exogenous and endogenous zinc ions in organisms.

[0169] The above is only a further description of one embodiment of the present invention, which cannot be used as a limitation of the claims. It should be understood that the present invention includes but is not limited to the above embodiment, and all modifications, replacements and applications of its structure belong to the protection scope of the independent claims of the present invention.

Claims

1. A pyrene-Schiff base fluorescent derivative, characterized in that: The structural formula of the pyrene-Schiff base fluorescent derivative is as follows: Wherein, R is a substituted or unsubstituted o-hydroxy aromatic aldehyde, and the substituted or unsubstituted o-hydroxy aromatic aldehyde is one of the following structural formulas: In the above structural formula: R1, R2, R3 and R4 can be independently selected from hydrogen, hydroxyl, alkyl, alkoxy, halogen, substituted or unsubstituted C6~C 60 Aromatic ring group, substituted or unsubstituted C3~C 60 One of the aromatic heterocyclic groups.

2. The method for preparing the pyrene-Schiff base fluorescent derivative according to claim 1, characterized in that: The following steps are involved: (1) 1-bromopyrene, 2-aminopyridine-5-boric acid pinacol ester, potassium carbonate aqueous solution, tetrakis(triphenylphosphine)palladium and 1,4-dioxane are mixed, heated and stirred under reflux, and then the reaction solution is mixed with an aqueous solution containing saturated sodium chloride, stirred and then allowed to stand, filtered, washed with water and the filter cake is dried, separated and purified by column chromatography using an eluent, and the organic solvent is removed by rotary evaporation to obtain an intermediate Py-NH2; (2) After mixing the intermediate Py-NH2, the raw material X, glacial acetic acid and anhydrous ethanol, the mixture is heated, stirred and refluxed, and filtered while hot, and then washed with anhydrous ethanol to obtain a crude target product, and the crude target product is dissolved by heating with anhydrous ethanol, and filtered while hot, and the reaction system is purified to obtain a target product, a pyrene Schiff base fluorescent derivative; The structural formula of the raw material X is R=O, R is a substituted or unsubstituted o-hydroxy aromatic aldehyde, and the substituted or unsubstituted o-hydroxy aromatic aldehyde is one of the following structural formulas: In the above structural formula: R1, R2, R3 and R4 can be independently selected from hydrogen, hydroxyl, alkyl, alkoxy, halogen, substituted or unsubstituted C6~C 60 Aromatic ring group, substituted or unsubstituted C3~C 60 One of the aromatic heterocyclic groups.

3. The method for preparing the pyrene-Schiff base fluorescent derivative according to claim 2, characterized in that: In step (1), the molar ratio of 1-bromopyrene, 2-aminopyridine-5-boric acid pinacol ester, potassium carbonate aqueous solution, tetrakis(triphenylphosphine)palladium and 1,4-dioxane is 1:(1.1-1.3):(3-5):(0.05-0.15):(80-120).

4. The method for preparing the pyrene-Schiff base fluorescent derivative according to claim 2, characterized in that: In step (1), the eluent for column chromatography separation is a dichloromethane-methanol system, and the volume ratio of the two is (90-110):

1.

5. The method for preparing the pyrene-Schiff base fluorescent derivative according to claim 2, characterized in that: In step (1), the eluent for column chromatography separation is a dichloromethane-methanol system, and the volume ratio of the two is 100:

1.

6. The method for preparing the pyrene-Schiff base fluorescent derivative according to claim 2, characterized in that: In step (2), the molar ratio of the intermediate Py-NH2, the raw material X and anhydrous ethanol is 1:(1.1-1.3):(20-30).

7. The method for preparing a pyrene-Schiff base fluorescent derivative according to claim 2, characterized in that: In step (2), the molar ratio of the intermediate Py-NH2, the raw material X and anhydrous ethanol is 1:1.1:

20.

8. A method for preparing a fluorescent probe using the pyrene Schiff base fluorescent derivative according to claim 1, characterized in that: The following steps are involved: (1) Dissolve the pyrene Schiff base fluorescent derivative in an organic solvent to prepare 1×10 -2 mol / L~5×10 -2 mol / L fluorescent probe stock solution; (2) Dilute the fluorescent probe stock solution to 1×10 -5 mol / L~5×10 -5 mol / L solution, shake well, and place the reaction solution in a dark environment at room temperature for 2 hours to obtain a fluorescent probe.

9. A fluorescent probe prepared by the method according to claim 8.

10. Use of the fluorescent probe according to claim 9 in exogenous / endogenous zinc ion fluorescence imaging, characterized in that: The object to be treated is incubated with the fluorescent probe in the dark for 30 to 50 minutes, and live cell imaging is performed using a laser confocal fluorescence microscope.

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