A colorimetric fluorescent probe for copper ions and synthesis and application thereof

Abstract

The application discloses a colorimetric copper ion fluorescent probe and synthesis and application thereof, which has a fluorophore and a copper ion recognition group, wherein the copper ion recognition group is one of commercial drugs of norfloxacin, levofloxacin, moxifloxacin and gatifloxacin. The copper ion fluorescent probe can be used for qualitative and colorimetric detection of copper ions, and is especially used in the fields of solution / cell / tissue / living body imaging, biological labeling or sensing.

Description

Technical Field

[0001] This invention belongs to the field of bioanalytical detection technology, specifically relating to a colorimetric copper ion fluorescent probe and its synthesis and application. Background Technology

[0002] Copper ions are the second most abundant trace element in living organisms and play a vital role in physiological processes. Studies have shown that the normal functioning of dozens of enzymes and proteins in the body is closely related to copper ions. Cells and tissues require an appropriate concentration of copper ions to maintain normal cellular metabolism and tissue function. Abnormal copper ion concentrations are associated with various diseases, including Menke syndrome and Alzheimer's disease. Therefore, monitoring internal copper ion levels is of great significance. Existing technologies have developed various methods for detecting copper ion levels, including spectrophotometry, atomic absorption spectrometry, inductively coupled plasma mass spectrometry, and optical imaging. Among these, optical imaging features high sensitivity, high spatiotemporal resolution, and non-invasive imaging, making it more suitable for in-situ real-time imaging in vivo.

[0003] Currently, several copper ion fluorescent probes have been reported, including coordination probes and reactive probes. Coordination probes are susceptible to interference from other transition metal ions, resulting in poor selectivity. Reactive probes are specific, thus exhibiting high selectivity for copper ions. Colorimetric probes allow for simple identification by visually observing color changes, offering advantages such as being unrestricted by equipment and easy to operate. Among the reported copper ion fluorescent probes, reactive colorimetric probes are relatively rare. (Zile Zhou, Shengyou Chen, Yinghui Huang, et al. Simultaneous visualization and quantification of copper(II)ions in Alzheimer's disease by anear-infrared fluorescence probe, Biosensors and Bioelectronics, 2022, 198, 113858; Soma Sarkar, Swapnadip Roy, Anindita Sikdar, et al. Apyrene-based simplebut highly selective fluorescence sensor for Cu 2+ions via a static excimermechanism,Analyst,2013,6,21;Keyin Liu,Huiming Shang,Fangfang Meng,et al.Anovel near-infrared fluorescent platform with good photostability and theapplication for a reaction-based probe in living cells,Talanta,2016,147,193-198;Yin Chen,Zhiqing Long,Chengcheng Wang,et al.A lysosome-targeted near-infrared fluorescent probe for cell imaging ofCu 2+,Dyes and Pigments,2022,204,110472;Yi Liu,Qianqian Su,Min Chen,et al.Near-Infrared UpconversionChemodosimeter for In Vivo Detection ofCu 2+in Wilson Disease,Adv.Mater.2016,28,6625–6630.)(Chunxia Li,Jinliang Liu,Sylvie Alonso,et al.Upconversionnanoparticles for sensitive and in-depth detection ofCu2+ions,Nanoscale,2012,4,6065;Zhu,Anwei,Qu,Qiang,Shao,Xiangling,et al.Carbon-Dot-Based Dual-EmissionNanohybrid Produces a Ratiometric Fluorescent Sensor for In Vivo Imaging ofCellular Copper Ions,Angewandte Chemie,International Edition,2012,51,7185-7189;Hou,Jinjie,Jia,Pei,Yang,Kairong,et al.Fluorescence and ColorimetricDual-Mode Ratiometric Sensor Based on Zr-Tetraphenylporphyrin TetrasulfonicAcid Hydrate Metal-Organic Frameworks for Visual Detection of Copper Ions,ACSApplied Materials&Interfaces,2022,14,13848-13857;Zhang,Wei,Zhang,Yu,Liu,Xin,et al.Ratiometric fluorescence and colorimetric dual-mode sensing platform based on carbon dots for detecting copper(II)ions and D-penicillamine, Analytical and Bioanalytical Chemistry, 2022, 414, 1651-1662.). . Summary of the Invention

[0004] Technical problems to be solved:

[0005] This application addresses the shortcomings of existing technologies by solving the problems of poor selectivity and the paramagnetic quenching of fluorescence by divalent copper ions in the recognition process of current coordination probes, which are easily interfered with by other transition metal ions. (Lin.Xu, Yufang.Xu, Weiping, Zhu, et al. Versatile trifunctional chemosensor of rhodamine derivative for Zn) 2+ Cu 2+ and His / Cys in aqueous solution and living cells,Org.Biomol.Chem.,2011,9,8284;G.He,X.Hua,N.Yang,et al,Synthesis and application of a “turn on” fluorescent probe for glutathione based on a coppercomplex of coumarin hydrazide Schiff base derivative,Bioorganic Chemistry,2019,91,103176;N.Ahmed,W.Zareen,D.Zhang,et al,Coumarin-Based ReversibleFluorescent Probe for Selective Detection ofCu 2+in Living Cells, Journal of Fluorescence, 2020, 30, 1171–1179; L. Zhao, K. Chen, K. Xie, et al, Abenzothiazole-based “on-off” fluorescence probe for the specific detection of Cu 2+ This paper addresses technical issues such as the application of copper ions in solution and living cells (Dyes and Pigments, 2023, 210, 110943). It provides a colorimetric fluorescent probe for copper ions, its synthesis and application. The probe recognizes copper ions through two methods: changes in probe color and changes in fluorescence spectrum.

[0006] Technical solution:

[0007] To achieve the above objectives, this application provides the following technical solution:

[0008] A colorimetric copper ion fluorescent probe has a fluorophore and a copper ion recognition group, wherein the copper ion recognition group is one or more of the commercial drugs norfloxacin, levofloxacin, moxifloxacin, and gatifloxacin.

[0009] Furthermore, the fluorophore is selected from one or more of the following: cyanine dyes, rhodamines, coumarins, fluoroboron pyrroles, fluoresceins, naphthalimides, and acridine dyes.

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

[0011] Furthermore, the cyanobacterium fluorophore is a heptamethylcyanobacterium fluorophore.

[0012] Furthermore, the heptamethrin fluorophore is an IR780 fluorophore.

[0013] Furthermore, the colorimetric copper ion fluorescent probe, abbreviated as IR-NFX, has the following structural formula:

[0014]

[0015] Furthermore, the colorimetric copper ion fluorescent probe binds to metal anions.

[0016] Furthermore, the anion is selected from Cl-, Br-, etc. - I-, NO3 - or PF4 - .

[0017] Applications of colorimetric copper ion fluorescent probes in the fields of solution, cell, tissue, imaging, biolabeling, or sensing.

[0018] This application also discloses a method for synthesizing a colorimetric copper ion fluorescent probe, the specific steps of which are as follows:

[0019] Step 1: Dissolve 0.15 mmol IR780 and 0.6 mmol norfloxacin in 5 mL of anhydrous DMF, and heat to 80 °C.

[0020] The reaction was refluxed for 5.5 hours under nitrogen protection, and the reaction progress was monitored by TLC.

[0021] Step 2: After the reaction was complete, the solvent was removed under reduced pressure, and the mixture was purified by silica gel column chromatography with CH2Cl2 / CH3OH (1 / 80, v / v) as the eluent, yielding 77.7 mg of the blue solid compound IR-NFX, with a yield of 44.63%. The reaction formula is as follows:

[0022] The principle of the above-mentioned colorimetric copper ion fluorescent probe and its synthesis and application is as follows: the lipid-soluble positive charge of cyanide dye has mitochondrial targeting properties, which can enable the fluorescent probe to target mitochondria. Alternatively, existing fluorophores can be modified with groups carrying lipid-soluble positive charges to achieve mitochondrial targeting. IR780 can target tumor cells, thereby causing drugs conjugated with it to accumulate in tumor cells, which has the potential to achieve integrated diagnosis and treatment.

[0023] Beneficial effects:

[0024] This application provides a colorimetric copper ion fluorescent probe, its synthesis and application, which has the following advantages compared with the prior art:

[0025] 1. The copper ion fluorescent probe of this invention has a maximum absorption peak around 707 nm and an emission peak at 615 nm. After the addition of copper ions, the maximum absorption peak shifts to around 540 nm, and the fluorescence emission peak shifts to 565 nm. The fluorescence emission peak shifts by nearly 50 nm and the ultraviolet absorption peak shifts by nearly 170 nm after the addition of copper ions. Within the copper ion concentration range of 0-60 μM, there is a good linear relationship between the fluorescence intensity and ultraviolet absorbance and the change in copper ion concentration (see appendix). Figure 2 This facilitates the quantitative detection of changes in copper ion content; simultaneously, the solution is emerald green before the addition of copper ions, turning blue-purple afterward, which is beneficial for detecting copper ions using colorimetric methods (see attached). Figure 3 ).

[0026] 2. The copper ion fluorescent probe described in this invention exhibits high selectivity and sensitivity for copper ion recognition. The recognition process is unaffected by common biological species under physiological pH conditions. Zinc ions and cobalt ions, which easily interfere with copper ion recognition, do not cause significant changes in the probe's fluorescence emission and ultraviolet absorption (Lin. Xu, Yufang. Xu, Weiping, Zhu, et al. Versatile trifunctional chemosensor of rhodamine derivative for Zn). 2+ Cu 2+ and His / Cys in aqueous solution and living cells, Org. Biomol. Chem., 2011, 9, 8284; G. He, Attached Figure Description

[0027] Figure 1 The fluorescence emission spectrum of the copper ion fluorescent probe IR-NFX described in this application in response to copper ions, wherein (a) Cu is added to the probe IR-NFX. 2+ Post-fluorescence spectrum, (b) fluorescence intensity at 568 nm as Cu 2+ Concentration change graph;

[0028] Figure 2 The UV-Vis absorption spectrum of the copper ion fluorescent probe IR-NFX described in this application in response to copper ions, wherein (a) Cu is added to the probe IR-NFX. 2+ The UV spectrum after (b) shows the ratio of UV absorption intensity at 706 nm to 542 nm as Cu 2+ Concentration change graph;

[0029] Figure 3 This is a color comparison image of the solution before and after adding copper ions to the copper ion fluorescent probe IR-NFX described in this application;

[0030] Figure 4 The fluorescence (a) and ultraviolet (b) spectral responses of the copper ion fluorescent probe IR-NFX described in this application to common species are shown.

[0031] Figure 5 The fluorescence (a) and ultraviolet (b) spectra of the copper ion fluorescent probe IR-NFX described in this application in the pH range of 5.5–8.5;

[0032] Figure 6 The graphs show the changes in fluorescence emission (a) and ultraviolet absorption (b) spectra of the copper ion fluorescent probe IR-NFX described in this application after the addition of copper ions, or after the addition of copper ions and EDTA. Detailed Implementation

[0033] To further illustrate this application, the following detailed description is provided in conjunction with embodiments.

[0034] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operation processes. However, the scope of protection of the present invention is not limited to the following examples.

[0035] IR780 (CAS No. 207399-07-3, purchased from Beijing Innocare Technology Co., Ltd.)

[0036] Example 1:

[0037] Preparation of a compound IR-NFX

[0038]

[0039] IR780 (100 mg, 0.15 mmol) and norfloxacin (191.6 mg, 0.6 mmol) were dissolved in 5 mL of anhydrous DMF, heated to 80 °C, and refluxed under nitrogen for 5.5 h. The reaction progress was monitored by TLC. After the reaction was complete, the solvent was removed under reduced pressure, and the mixture was purified by silica gel column chromatography with CH2Cl2 / CH3OH (1 / 80, v / v) as the eluent to give 77.7 mg of the blue solid compound IR-NFX, in a yield of 44.63%.

[0040] 1H NMR(400MHz,Chloroform-d,δ,ppm)15.26(s,1H),8.73(s,1H),8.12(d,J=13.3H z,1H),7.76(d,J=13.5Hz,2H),7.35–7.27(m,4H),7.14(t,J=7.5Hz,2H),6.97(d ,J=7.9Hz,2H),5.83(d,J=13.4Hz,2H),3.98–3.83(m,12H),2.49(t,J=6.5Hz,4H ),1.91–1.84(m,6H),1.69(s,12H),1.62(d,J=7.5Hz,7H),1.07(t,J=7.4Hz,6H). 13 C NMR(101MHz,Chloroform-d,δ,ppm)176.98,172.65,169.67,167.56,147.31,142.64,142.06,140.30,137.74,128.48,124 .52,124.00,122.23,109.49,107.06,96.65,54.87,52.50,51.60,48.41,45.32,29.25,25.04,21.77,20.45,15.07,11.80.

[0041] Example 2:

[0042] A probe IR-NFX responds to the fluorescence and ultraviolet spectra of copper ions.

[0043] Copper nitrate solution was added to a HEPES solution containing 20 μM probe IR-NFX (pH 7.4, 10 mM, 30% acetonitrile, v / v) to bring the copper ion concentration in the solution to the range of 0 to 140 μM. Data were collected after the spectrum stabilized. Figure 1 As can be seen, the fluorescence emission peak of the probe IR-NFX is around 615 nm. Once Cu 2+ The fluorescence emission of the probe is completely shifted to around 568 nm, and the intensity of this emission peak increases with Cu. 2+ The effect increases with increasing concentration, and a clear linear relationship exists between the two within the range of 0–70 μM copper ion concentration. Figure 2 As can be seen, the probe IR-NFX has the strongest absorption peak near 707 nm, at which point the solution color is emerald green. 2+ After the addition, the intensity of the absorption peak decreased, and a new absorption peak appeared near 542 nm, while the solution color changed to blue-purple. Figure 3 Similarly, Abs in the 0–70 μM range 707nm A clear linear relationship exists between the concentration of copper ions and the concentration of copper ions.

[0044] Example 3:

[0045] The fluorescence and ultraviolet spectral responses of a probe IR-NFX to common species of various life forms

[0046] 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 IR-NFX. Data were collected after the spectra stabilized. Species concentration: 2 mM Na + K + Mg 2+ Ca 2+ 50μM Zn 2+ Co 2+ Cu 2+ Fe 3+ Fe 2+ 2mM Cl - 1mM F - I - HPO4 2- HCO3 - 200μM HS - ,1mM GSH,200μM Cys. Figure 4 It is evident that the fluorescence and UV spectra of the probe IR-NFX did not change significantly after the addition of other species, while the fluorescence emission and UV absorption changed significantly only after the addition of copper ions.

[0047] Example 4

[0048] The effect of pH on the fluorescence emission and UV absorption of the probe IR-NFX

[0049] HCl or NaOH was added to a HEPES solution containing 20 μM probe IR-NFX (10 mM, 30% acetonitrile, v / v) to adjust the pH to the target value. Data were collected after the spectrum stabilized. Figure 5 It is evident that the fluorescence emission and ultraviolet absorption of the probe IR-NFX are almost unaffected by pH changes within the physiological pH range.

[0050] Example 5

[0051] Cu was added to a probe IR-NFX 2+ Changes in fluorescence and UV spectra after EDTA

[0052] Add 200 μM Cu to a HEPES solution (10 mM, 30% acetonitrile, v / v) containing 20 μM probe IR-NFX. 2+ back, Figure 6 (a) The emission peak of the visible probe shifts from 615 nm to around 565 nm. Cu is then added to the above solution.2+ The chelating agent EDTA (400 μM) was used. After the signal stabilized, the fluorescence intensity at 565 nm was significantly increased. Figure 6 (b) It can be seen that 200 μM Cu was added. 2+ Subsequently, the strongest absorption peak of the probe near 707 nm decreased, and Cu was further added to the above solution. 2+ The absorption peak showed no significant change when the chelating agent EDTA (400 μM) was used. This indicates that the probe reacts with Cu. 2+ There is no reversible coordination between them, Cu 2+ The probe underwent a chemical reaction after being added.

[0053] The above description of this application is exemplary. It should be noted that, without departing from the core of this application, any simple variations, modifications, or other equivalent substitutions that can be made by those skilled in the art without creative effort fall within the protection scope of this application.

Claims

1. A colorimetric copper ion fluorescent probe, characterized in that: The colorimetric copper ion fluorescent probe, abbreviated as IR-NFX, has the following structural formula: .

2. The colorimetric copper ion fluorescent probe according to claim 1, characterized in that: The colorimetric copper ion fluorescent probe binds to metal anions.

3. The colorimetric copper ion fluorescent probe according to claim 2, characterized in that: The metal anion is selected from Cl. - ,Br - I - NO3 - or PF4 - .

4. A method for synthesizing a colorimetric copper ion fluorescent probe according to any one of claims 1-3, characterized in that, The reaction formula is as follows: .

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