Coordination type copper ion fluorescent probe as well as synthesis method and application thereof

Abstract

The invention discloses a coordination type copper ion fluorescent probe and a synthesis method and application thereof, the coordination type copper ion fluorescent probe has a fluorophore and a copper ion recognition group, and the copper ion recognition group is one of quinolone antibacterial drugs such as enoxacin and ciprofloxacin; the fluorescent probe shows copper ion selectivity, can be used for qualitative detection of copper ions, and is particularly applied to the fields of cell / tissue / living body imaging, biomarker or sensing.

Description

Technical Field

[0001] The invention belongs to the technical field of detection and imaging, and in particular relates to a coordination-type copper ion fluorescent probe and a synthesis method and application thereof. Background Art

[0002] Copper ions are essential trace elements in living organisms and play an important role in physiological processes. Studies have found that copper has a dual role in human health. Normal levels of copper can maintain the normal functioning of hematopoiesis and the central nervous system; when there is an excess of copper in the body, abdominal distension, hepatosplenomegaly, ascites, death and other adverse conditions may occur. Therefore, copper ion content is an important parameter affecting the body. When copper ion metabolism is unbalanced, it can lead to serious diseases such as cancer, Alzheimer's disease, Menkes syndrome and Wilson's disease. Methods for detecting copper ions include atomic absorption spectrometry, atomic fluorescence spectrometry, chemical titration, inductively coupled plasma, electrochemical analysis and chromatography. At present, most coordination probes are easily interfered by other transition metal ions during the recognition process, and have the problem of low selectivity (Hao Fang, Peng-Cheng Huang, Fang-Ying Wu, et al. A highly sensitive fluorescent probe with different responses to Cu 2+ and Zn 2+ .Spectrochimica Acta Part A:Molecularand Biomolecular Spectroscopy,2019,214:233-238;Bhavana G.Gowda,MuzaffarIqbal,ShivaPrasad Kollur,et al.A "Turn-Off" Pyrene-Based Ligand as aFluorescent Sensor for the Detection of Cu 2+ and Fe 2+Ions:Synthesis andApplication in Real Water Samples,Logic Gate Construction,and Bio-Imaging.Chemosensors,2024,12(6),91;SellamuthuAnbu,Anup Paul,KalpanaSurendranath,et al.Naphthalimide-phenanthroimidazole incorporated new fluorescent sensor for “turn-on” Cu 2+ detection in living cancer cells. Journal of Inorganic Biochemistry, 2021, 220: 111466; Sellamuthu Anbu, Anup Paul, Kalpana Surendranath, et al. Naphthalimide-phenanthroimidazole incorporated new fluorescent sensor for “turn-on” Cu 2+ detection in living cancer cells.Journal of Inorganic Biochemistry, 2021, 220:111466.); and the paramagnetic fluorescence quenching of divalent copper ions, resulting in reduced detection sensitivity (Xiaoling Niu, Hong Zhang, Xiaojing Wu, et al. An AIE-active "turn-off" fluorescent sensor for highly selective and sensitive detection of Cu 2+ ions.Journal ofMolecular Structure,2022,1264:133294; ​​ShenglingLi,Duanlin Cao,Xianjiao Meng,et al.A novel fluorescent chemosensor based on coumarin and quinolinyl-benzothiazole for sequential recognition ofCu 2+and PPiand its applicability in live cell imaging. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 2020, 230: 118022.), the traditional method is limited by cumbersome sample pretreatment, time-consuming analysis, inability to detect in real time in vivo and high-cost instruments, and is difficult to be widely used in life detection. Therefore, a research method that can detect copper ions in cells or in vivo in real time and non-destructively is needed. The fluorescent probe method is a detection method that has attracted much attention in recent years. It has the advantages of simple operation, rapid detection, good selectivity, high sensitivity, and the ability to detect in vivo non-destructively, so it is suitable for detecting copper ions in living bodies.

[0003] At present, many examples of copper ion fluorescent probes have been reported, including coordination probes and reaction probes. The mechanism of action of coordination probes is mainly through the coordination of copper ions with N, O, S and other atoms with strong coordination effects in the probe, changing the PET, ICT and other effects in the probe, thereby causing changes in the intensity of the fluorescence signal or shifting the emission peak. The coordination effect is generally a reversible and rapid process, so this type of probe is expected to achieve the detection of Cu in vivo. 2+ Ahmed N, Zareen W, Zhang D, et al. Coumarin-based reversible fluorescent probe for selective detection of Cu 2+ in living cells.Journal of Fluorescence,2020,30:1171-1179;Wu Y,Li C,Li Y,et al.Development of a simple pyrene-basedratiometric fluorescent chemosensor for copper ion in living cells.Sensors&Actuators:B.Chemical,2016,222(C):1226-1232;Kanokorn W,Utt E, Jintana M, et al. A highly sensitive and selective rhodamine-semicarbazide based fluorescent sensor for Cu 2+detection in real water samples and fluorescence bioimaging inHepG2 cells. Talanta, 2024, 270:125530.). Summary of the invention

[0004] Technical issues solved:

[0005] The present application aims at the deficiencies of the prior art and solves the problems that most of the current coordination probes are easily interfered by other transition metal ions during the recognition process and have low selectivity, as well as the paramagnetic fluorescence quenching of divalent copper ions, which leads to reduced detection sensitivity. Traditional methods are limited by technical problems such as cumbersome sample pretreatment, time-consuming analysis, inability to detect in real time in vivo and high-cost instruments, making them difficult to be widely used in life detection. A coordination copper ion fluorescent probe and its synthesis method and application are provided. The probe can recognize copper ions through two ways: changes in the color of the probe and changes in the fluorescence spectrum.

[0006] Technical solution:

[0007] To achieve the above objectives, this application is implemented through the following technical solutions:

[0008] A coordination copper ion fluorescent probe comprises a fluorophore and a copper ion recognition group. The copper ion recognition group is a quinolone antibiotic. The quinolone antibiotic is enoxacin and / or ciprofloxacin.

[0009] Furthermore, the fluorophore is one or more of fluorescein, coumarin, rhodamine, naphthylimide, boropyrrole, and cyanine dye fluorescent groups.

[0010] Furthermore, the cyanine dye fluorophore is a cyanine dye fluorophore.

[0011] Furthermore, the cyanine-based fluorophore is a heptamethine cyanine-based fluorophore.

[0012] Furthermore, the heptamethine cyanine fluorophore is an IR780 fluorophore.

[0013] Furthermore, the coordination copper ion fluorescent probe is IR780-ENO, and the structural formula is as follows:

[0014]

[0015] A method for synthesizing a coordinated copper ion fluorescent probe comprises the following steps:

[0016] Step 1: 0.15 mmol IR780 and 0.6 mmol enoxacin were dissolved in 5 mL DMF, heated to 80 °C, and refluxed for 4 hours under nitrogen protection. The reaction progress was monitored by TLC.

[0017] Step 2: After the reaction is completed, the solvent is removed under reduced pressure, and the product is separated and purified using a silica gel column, with the eluent being CH2Cl2 / CH3OH (40 / 1, v / v), to obtain a blue solid compound IR780-ENO, with the following reaction formula:

[0018]

[0019] The present application also discloses the application of the coordinated copper ion fluorescent probe in the field of solution, cell, tissue and living body imaging.

[0020] Furthermore, the coordinated copper ion fluorescent probe is combined with anions.

[0021] Further, the anion is selected from I - or PO4 3- .

[0022] Principle explanation: The fat-soluble positive charge of Sichuan cyanine dye has mitochondrial targeting properties, which can target fluorescent probes to mitochondria. Existing fluorophores can also be modified with groups such as triphenylphosphine groups to achieve mitochondrial targeting.

[0023] Beneficial effects:

[0024] The present application provides a coordination copper ion fluorescent probe and its synthesis method and application, which have the following beneficial effects compared with the prior art:

[0025] 1. The maximum emission peak of the copper ion fluorescent probe of the present invention is around 558nm, and the fluorescence emission intensity is significantly enhanced after the addition of copper ions;

[0026] 2. The maximum absorption peak is near 767nm. After the addition of copper ions, the maximum absorption peak is observed to shift to near 547nm, and the probe solution changes from emerald blue to light purple red;

[0027] 3. In the range of 0-20μM copper ion concentration, there is a good linear relationship between fluorescence intensity and ultraviolet absorbance and copper ion concentration, which is conducive to qualitative and semi-quantitative detection of changes in copper ion content;

[0028] 4. The copper ion fluorescent probe described in the present invention has the characteristics of high selectivity and high sensitivity in copper ion recognition, and the recognition process is not interfered by common species of living organisms under physiological pH conditions. Among them, iron ions, zinc ions, etc. that easily interfere with copper ion recognition do not cause obvious changes in probe fluorescence emission and ultraviolet absorption, thereby realizing copper ion imaging in living lung cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is the fluorescence emission spectrum of the copper ion fluorescent probe IR780-ENO in this application in response to copper ions, wherein a is the fluorescence emission spectrum of the copper ion fluorescent probe IR780-ENO in response to copper ions. 2+ Fluorescence spectrum of Cu 2+ Concentration variation graph;

[0030] Figure 2 is the UV-visible absorption spectrum of the copper ion fluorescent probe IR780-ENO described in this application in response to copper ions, wherein a is the amount of Cu added to the probe IR780-ENO. 2+ The UV spectrum after Cu 2+ Concentration variation relationship diagram;

[0031] Figure 3 This is a diagram showing the color change of the solution before and after adding copper ions to the copper ion fluorescent probe IR780-ENO described in this application;

[0032] Figure 4 The fluorescence and ultraviolet spectrum response diagrams of the copper ion fluorescent probe IR780-ENO described in this application to common species of life, wherein a is the fluorescence spectrum response diagram and b is the ultraviolet spectrum response diagram;

[0033] Figure 5 The fluorescence and ultraviolet spectra of the copper ion fluorescent probe IR780-ENO described in this application in the range of pH = 4.54-8.50, wherein a is the fluorescence spectrum and b is the ultraviolet spectrum;

[0034] Figure 6 This is a test diagram of the reversibility of the fluorescent probe IR780-ENO in response to copper ions described in this application, wherein a is a fluorescence emission spectrum diagram, and b is a UV absorption spectrum diagram;

[0035] Figure 7 This is a test diagram of the coordination ratio of the fluorescent probe IR780-ENO and copper ions described in this application, wherein a is a diagram of the change of the ultraviolet absorption spectrum, and b is a Job's plot curve diagram;

[0036] Figure 8 This is a microscopic imaging diagram of the fluorescent probe IR780-ENO described in this application on copper ions in A549 cells, wherein the left figure is a test diagram of the response of A549 cells to exogenous copper ions, and the right figure is a bar graph of the corresponding average fluorescence intensity in the cells. DETAILED DESCRIPTION

[0037] In order to further illustrate the present invention, the present invention is further described in detail below in conjunction with embodiments.

[0038] The following is a detailed description of an embodiment of the present invention. This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and a specific operation process are given, but the protection scope of the present invention is not limited to the following examples.

[0039] IR780 (cas number 207399-07-3) was purchased from Beijing Inokai Technology Co., Ltd.

[0040] Example 1

[0041] A method for synthesizing a coordinated copper ion fluorescent probe IR780-ENO specifically comprises the following steps:

[0042] Step 1: 0.15mmol IR780100mg and 0.6mmol enoxacin192.19mg were dissolved in 5mL DMF, heated to 80°C, and refluxed under nitrogen for 4 hours. The reaction progress was monitored by TLC.

[0043] Step 2: After the reaction is completed, the solvent is removed under reduced pressure, and the product is separated and purified using a silica gel column, with the eluent being CH2Cl2 / CH3OH (40 / 1, v / v).

[0044] The blue solid compound IR780-ENO is obtained, and the reaction formula is as follows:

[0045]

[0046] 1 H NMR (400MHz, CDCl3) δ15.04(s,1H),8.70(s,1H),8.15(d,J=12.9Hz,1H),7.80(d,J=13.4Hz, 2H),7.33(dd,J=16.2,8.5Hz,4H),7.15(t,J=7.5Hz,2H),7.01(d,J=8.0Hz,2H),5.93(d,J=13 .4Hz,2H),4.59–4.48(m,2H),4.27–4.16(m,4H),4.04–3.84(m,8H),2.55(t,J=6.5Hz,4H),1. 88(d,J=7.1Hz,6H),1.68(s,15H),1.54(t,J=7.1Hz,4H),1.25(s,2H),1.07(t,J=7.4Hz,6H).

[0047] 13C NMR (101MHz, CDCl3) δ177.00,171.32,169.57,166.83,146.65,145.05,142.68,141.52,140.26,125.49,124.06 ,122.14,109.73,109.22,97.48,54.22,48.85,48.43,48.36,45.52,29.04,25.37,21.65,20.51,15.24,11.80.

[0048] Example 2

[0049] Fluorescence and ultraviolet spectrum response of a coordinated copper ion fluorescent probe R780-ENO to copper ions:

[0050] Copper nitrate solution was added to a HEPES solution (pH 7.4, 10 mM, 30% acetonitrile, v / v) containing 20 μM probe IR780-ENO to adjust the copper ion concentration in the solution to the range of 0 to 20 μM. Data were collected after the spectrum was stable. Figure 1 It can be seen that the fluorescence emission peak of the probe IR780-ENO is around 558nm. 2+ After that, a slight red shift was observed in the fluorescence emission peak, and the strongest emission peak appeared near 570nm. 2+ The effect of copper ion on the effect of copper ion was enhanced with the increase of copper ion concentration, and there was an obvious linear relationship between the two in the range of copper ion concentration of 0-20μM. Figure 2 It can be seen that the probe IR780-ENO has the strongest absorption peak near 767nm, and the solution color is emerald blue at this time. 2+ After the addition, the absorption peak intensity decreased, and a new absorption peak appeared near 547nm. The color of the solution changed with the change of Cu 2+ As the concentration increases, it gradually transitions from jade blue to light purple red ( Figure 3 ). Also in the range of 0-20 μM Abs 763nm / Abs 547nm There is an obvious linear relationship between the concentration of copper ions.

[0051] Example 3

[0052] Fluorescence and ultraviolet spectrum responses of a coordinated copper ion fluorescent probe IR780-ENO to common species of various living organisms:

[0053] Common species of various life forms were added to a HEPES solution (pH 7.4, 10 mM, 30% acetonitrile, v / v) containing 20 μM probe IR780-ENO, and data were collected after the spectrum was stable. Concentration of each species: 1 mM GSH, 10 mM Na + , 10mM K+ , 10mMCa 2+ 、10mM Mg 2+ , 200μM H2O2, 100μM Cys, 10μM Zn 2+ , 50μM Cu 2+ , 10 μM Fe 2+ , 10 μM Fe 3+ and 10 μM Na2S. Figure 4 It can be seen that the fluorescence and ultraviolet spectra of the probe IR780-ENO did not change significantly after the addition of other species, and only the fluorescence emission and ultraviolet absorption changed significantly after the addition of copper ions.

[0054] Example 4

[0055] Effect of pH on fluorescence emission and ultraviolet absorption of coordinated copper ion fluorescent probe IR780-ENO:

[0056] HCl or NaOH was added to the HEPES solution (10 mM, 30% acetonitrile, v / v) containing 20 μM probe IR780-ENO to adjust the pH to the target value, and the data were collected after the spectrum stabilized. Figure 5 It can be seen that the fluorescence emission and ultraviolet absorption of the probe IR780-ENO within the physiological pH range are almost unaffected by pH changes.

[0057] Example 5

[0058] A coordinated copper ion fluorescent probe IR780-ENO and Cu 2+ Reversibility study:

[0059] 20 μM Cu was added to a HEPES solution (10 mM, 30% acetonitrile, v / v) containing 20 μM probe IR780-ENO. 2+ After the reaction reaches equilibrium, 200 μM EDTA is added. Figure 6 As shown in (a), the fluorescence emission is greatly enhanced after the addition of copper ions. After the addition of EDTA, the fluorescence intensity of the solution decreases to a level close to that of the pure probe IR780-ENO, proving that the probe IR780-ENO has a reversible coordination effect with copper ions.

[0060] Example 6

[0061] A coordinated copper ion fluorescent probe IR780-ENO and Cu 2+ The coordination ratio:

[0062] The probe IR780-ENO and Cu were immobilized in a HEPES buffer solution (10 mM, pH = 7.40) containing 30% acetonitrile (v / v). 2+Total concentration, prepare 10 equally spaced test solutions (Cu 2+ The mole fraction range is 0-0.9), and the ultraviolet absorption spectra of 10 solutions are detected. Figure 7 (b) shows that Cu 2+ When the molar fraction is 0.5, that is, the concentration of probe IR780-ENO is 2+ The inflection point appears when the concentration is the same, proving that the probe and Cu 2+ The coordination ratio is 1:1, which is consistent with the spectroscopic titration experiment.

[0063] Example 7

[0064] Fluorescence microscopy imaging of a coordinated copper ion fluorescent probe IR780-ENO in A549 lung cancer cells:

[0065] A549 lung cancer cell line was incubated with 10 μM probe IR780-ENO for 20 min or with 20 μM Cu 2+ After incubation for 30 min, 10 μM probe IR780-ENO was added and incubated for 20 min. Figure 8 The results showed that the probe IR780-ENO had good cell permeability and could successfully image A549 cells. The intracellular probe fluorescence signal was enhanced after the addition of exogenous copper ions, indicating that the probe IR780-ENO could indicate the changes of copper ions in A549 cells.

[0066] The above is an exemplary description of the present application. It should be noted that, without departing from the core of the present application, any simple deformation, modification or equivalent replacement that can be done by other technical personnel in this field without expending creative labor falls within the scope of protection of the present application.

Claims

1. A coordinated copper ion fluorescent probe, characterized in that: The coordination copper ion fluorescent probe has a fluorophore and a copper ion recognition group, the copper ion recognition group is a quinolone antibiotic, and the quinolone antibiotic is one or more of enoxacin, norfloxacin, and ciprofloxacin.

2. The coordinated copper ion fluorescent probe according to claim 1, characterized in that: The fluorophore is one or more of fluorescein, coumarin, rhodamine, naphthylimide, boropyrrole, and cyanine dye fluorescent groups.

3. The coordinated copper ion fluorescent probe according to claim 2, characterized in that: The cyanine dye fluorophore is a cyanine dye fluorophore.

4. The coordinated copper ion fluorescent probe according to claim 3, characterized in that: The cyanine-based fluorophore is a heptamethine cyanine-based fluorophore.

5. The coordinated copper ion fluorescent probe according to claim 4, characterized in that: The heptamethine cyanine fluorophore is an IR780 fluorophore.

6. The coordinated copper ion fluorescent probe according to claim 1, characterized in that: The coordination copper ion fluorescent probe is IR780-ENO, and the structural formula is as follows:

7. A method for synthesizing a coordinated copper ion fluorescent probe, characterized in that: The specific steps include: Step 1: 0.15mmol IR780 and 0.6mmol quinolone antibiotics were dissolved in 5mL DMF, heated to 80℃, and refluxed for 4 hours under nitrogen protection. The reaction progress was monitored by TLC. Step 2: After the reaction is completed, the solvent is removed under reduced pressure, and the product is separated and purified using a silica gel column, with the eluent being CH2Cl2 / CH3OH (40 / 1, v / v), to obtain a blue solid compound IR780-ENO, with the following reaction formula:

8. Use of the coordinated copper ion fluorescent probe according to any one of claims 1 to 6 in the field of solution, cell, tissue, and living body imaging.

9. The coordinated copper ion fluorescent probe according to claim 1, characterized in that: The coordinated copper ion fluorescent probe is combined with anions.

10. The coordinated copper ion fluorescent probe according to claim 9, characterized in that: The anion is selected from I - or PO4 3- .

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