Fluorescent probe based on hydroxyl free corrole as well as preparation method and application of fluorescent probe

Abstract

The invention discloses a fluorescent probe based on hydroxyl free corrole and a preparation method and application of the fluorescent probe. The fluorescent probe has single selectivity on Cu < 2 + > and can effectively detect Cu < 2 + >, and the detection limit is as low as 22 nmol / L. The problems of low sensitivity and poor selectivity in the technical field of copper ion fluorescent probe detection are effectively solved, meanwhile, through an innovative method and means, the purposes that the probe is good in selectivity and high in sensitivity, and copper ions in an environmental water body or living cells can be rapidly and efficiently recognized are achieved, the copper ion detection efficiency is improved, and the detection cost is reduced. And a remarkable promotion effect is brought to industry development.

Description

Technical Field

[0001] The invention relates to the technical field of analytical chemistry, and in particular to a fluorescent probe based on hydroxyl free corrole and a preparation method and application thereof. Background Art

[0002] In recent years, the design and development of new fluorescent chemical probes have attracted extensive attention due to their important applications in materials science, biomedical analysis, environmental science and analytical chemistry. Molecular probes are often useful tools for monitoring relevant analytes in vivo or in vitro. Reliable quantitative detection of pollutants and biological species in the environment can provide necessary information for the appropriate treatment of contaminated environments and biomedical materials. At present, organic molecular probes based on small molecules have broad development prospects due to their advantages such as low cost, easy operation and wide application range. Therefore, a large number of high-performance probes have been designed and developed for the detection of various important environmental and biomedical target analytes, such as metal ions, anions, small organic molecules, DNA, RNA, proteins, and even cells, bacteria and viruses. From this perspective, the development of fluorescent probes with good selectivity, excellent tolerance to complex detection environments, good stability and high repeatability is a current research hotspot.

[0003] Copper is widely present in soil, atmosphere, environmental water and organisms. It is also one of the indispensable trace elements for the human body and plays an important role in the basic physiological activities of living organisms. Excessive Cu produced by mining and smelting industries 2+ Waste is an important cause of environmental pollution. In addition, copper, as a cofactor of many enzymes related to energy metabolism and anti-oxidative stress, plays a key role in maintaining various physiological processes in organisms. The body's intake and metabolism of copper need to be maintained in a strict balance. Any imbalance may cause serious health problems. Many human diseases are related to copper deficiency and excess. In the human brain, copper is particularly important for the maintenance of neuronal function because it is involved in biological processes such as the synthesis of neurotransmitters and cellular respiration. At the same time, copper also plays a key role in the basic physiological functions of brain cells and plays an important role in neuronal signaling pathways. Therefore, ensuring that the human body consumes an appropriate amount of copper is essential for good health.

[0004] Currently, the prior art has reported many examples of copper ion fluorescent probes, including coordination probes and reaction probes. However, the recognition process of most coordination probes is easily interfered by other transition metal ions and has poor selectivity. Summary of the invention

[0005] The purpose of the present invention is to provide a fluorescent probe based on hydroxyl free corrole and a preparation method and application thereof, so as to solve the problems of low sensitivity and poor selectivity in the technical field of copper ion fluorescent probe detection.

[0006] To achieve the above-mentioned purpose, the present invention adopts the following technical solutions:

[0007] A fluorescent probe based on hydroxyl free corrole is used for selectively identifying copper ions. The structural formulas of the fluorescent probe based on hydroxyl free corrole are as follows:

[0008]

[0009] The present invention also provides a method for a fluorescent probe based on hydroxyl free corrole, wherein the general reaction formula of the method is:

[0010]

[0011] Specifically, the method comprises the following steps: weigh pentafluorophenyl dipyrrolidine and place it in a round-bottom flask, add salicylaldehyde or m-hydroxybenzaldehyde or p-hydroxybenzaldehyde, and dissolve it in dichloromethane (DCM); after stirring evenly, add trifluoroacetic acid (TFA), react at room temperature for a period of time, then add triethylamine (Et3N), and after the smoke disappears, add an appropriate amount of dichloromethane; then add 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ), stir and react at room temperature for a period of time; separate by chromatography on a 100-200 mesh coarse silica gel column, use dichloromethane as an eluent, collect substances with red fluorescence under 365nm ultraviolet light, and obtain a crude product by vacuum rotary evaporation; separate the crude product by chromatography on a 300-400 mesh fine silica gel column, use dichloromethane and n-hexane as eluents, collect red fluorescent substances, and obtain a purple solid by vacuum rotary evaporation.

[0012] Preferably, methyl chloride and n-hexane are used as the eluent in a ratio of 1:1.

[0013] The fluorescent probe based on hydroxyl free corrole of the present invention is used for Cu 2+ With single selectivity, it can effectively detect Cu 2+ , the detection limit is as low as 22nmol / L. It effectively solves the problems of low sensitivity and poor selectivity in the field of copper ion fluorescent probe detection technology. At the same time, the present invention achieves the goal of good probe selectivity, high sensitivity, and the ability to quickly and efficiently identify copper ions in environmental water or living cells through innovative methods and means, thereby improving the efficiency of copper ion detection and bringing significant promotion to the development of the industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The hydroxycorrole probe 1 of the present invention 1 H NMR spectra;

[0015] Figure 2 The hydroxycorrole probe 1 of the present invention 19 F NMR spectra;

[0016] Figure 3 is the HR-MS spectrum of hydroxycorrole probe 1;

[0017] Figure 4 is the hydroxycorrole probe 2 of the present invention 1 H NMR spectra;

[0018] Figure 5 is the hydroxycorrole probe 2 of the present invention 19 F NMR spectra;

[0019] Figure 6 is the HR-MS spectrum of hydroxycorrole probe 2;

[0020] Figure 7 is the hydroxycorrole probe 3 of the present invention 1 H NMR spectra;

[0021] Figure 8 is the hydroxycorrole probe 3 of the present invention 19 F NMR spectra;

[0022] Fig. 9 is the HR-MS spectrum of hydroxycorrole probe 3;

[0023] Fig.10 is the fluorescence spectrum of hydroxycorrole probe 1 under different metal ion and anion conditions;

[0024] Fig.11 Photos of hydroxycorrole probe 1 added with different metal ions and anions (under 365nm UV light);

[0025] Fig.12 is the fluorescence spectra of hydroxycorrole probe 2 under different metal ion and anion conditions;

[0026] Fig.13 Photos of hydroxycorrole probe 2 added with different metal ions and anions (under 365nm UV light);

[0027] Fig.14 is the fluorescence spectrum of hydroxycorrole probe 3 under different metal ion and anion conditions;

[0028] Fig.15 Photos of hydroxycorrole probe 3 added with different metal ions and anions (under 365nm UV light);

[0029] Fig.16 Different concentrations of Cu 2+ Fluorescence spectrum of hydroxycorrole probe 1 in the presence of;

[0030] Fig.17 Different concentrations of Cu 2+Linear fitting diagram of fluorescence intensity of hydroxycorrole probe 1 when it is present;

[0031] Fig.18 Different concentrations of Cu 2+ Fluorescence spectrum of hydroxycorrole probe 2 in the presence of;

[0032] Fig.19 Different concentrations of Cu 2+ Linear fitting diagram of fluorescence intensity of hydroxycorrole probe 2 when it is present;

[0033] Fig. 20 Different concentrations of Cu 2+ Fluorescence spectrum of hydroxycorrole probe 3 in the presence of;

[0034] Fig.21 Different concentrations of Cu 2+ Linear fitting plot of fluorescence intensity of hydroxycorrole probe 3 in the presence of DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the accompanying drawings in the embodiments of the present invention, but this does not constitute a limitation on the protection scope of the present invention.

[0036] (1) Preparation of probe

[0037] Hydroxyphenyl-substituted amphiphilic corrole derivatives were prepared and their structures were characterized by NMR:

[0038]

[0039] The synthetic route is as follows:

[0040]

[0041] Preparation method of o-hydroxycorrole probe (10-(2-hydroxyphenyl)-5,15-(pentafluorophenyl)corrole, 1): Weigh 1.2g of pentafluorophenyl dipyrrolidine and place it in a 250mL round-bottom flask, add 135μL of salicylaldehyde, and dissolve it in 60mL of dichloromethane (DCM). After stirring evenly, add 12μL of trifluoroacetic acid (TFA) and react at room temperature for 4.5h. Add 12μL of triethylamine (Et3N), and after the smoke disappears, add 40mL of dichloromethane. Then add 0.8g of 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) and stir at room temperature for 1h. Use 100-200 mesh coarse silica gel column chromatography to separate, use dichloromethane as eluent, collect the substance with red fluorescence under 365nm ultraviolet light, and evaporate under reduced pressure to obtain the crude product. The crude product was separated by 300-400 mesh fine silica gel column chromatography, using dichloromethane and n-hexane in a ratio of 1:1 as eluent, collecting the red fluorescent substance, and obtaining a purple solid after rotary evaporation under reduced pressure. The yield was 10%.

[0042] Preparation method of m-hydroxycorrole probe (10-(3-hydroxyphenyl)-5,15-(pentafluorophenyl)corrole, 2): Weigh 1.2g of pentafluorophenyl dipyrrolidine and place it in a 250mL round-bottom flask, add 0.1655g of m-hydroxybenzaldehyde, and dissolve it in 60mL of dichloromethane (DCM). After stirring evenly, add 12μL of trifluoroacetic acid (TFA) and react at room temperature for 4.5h. Add 12μL of triethylamine (Et3N), and after the smoke disappears, add 40mL of dichloromethane. Then add 0.8g of 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) and stir at room temperature for 1h. Use 100-200 mesh coarse silica gel column chromatography to separate, use dichloromethane as eluent, collect the substance with red fluorescence under 365nm ultraviolet light, and obtain the crude product by vacuum rotary evaporation. The crude product was separated by 300-400 mesh fine silica gel column chromatography, using dichloromethane and n-hexane in a ratio of 1:1 as eluent, collecting the red fluorescent substance, and obtaining a purple solid after rotary evaporation under reduced pressure. The yield was 12%.

[0043] Preparation method of p-hydroxycorrole probe (10-(4-hydroxyphenyl)-5,15-(pentafluorophenyl)corrole, 3): Weigh 1.2g of pentafluorophenyl dipyrrolidine and place it in a 250mL round-bottom flask, add 0.1655g of p-hydroxybenzaldehyde, and dissolve it in 60mL of dichloromethane (DCM). After stirring evenly, add 12μL of trifluoroacetic acid (TFA) and react at room temperature for 4.5h. Add 12μL of triethylamine (Et3N), and after the smoke disappears, add 40mL of dichloromethane. Then add 0.8g of 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) and stir at room temperature for 1h. Use 100-200 mesh coarse silica gel column chromatography to separate, use dichloromethane as the eluent, collect the substance with red fluorescence under 365nm ultraviolet light, and obtain the crude product by vacuum rotary evaporation. The crude product was separated by 300-400 mesh fine silica gel column chromatography, using dichloromethane and n-hexane in a ratio of 1:1 as eluent, collecting the red fluorescent substance, and obtaining a purple solid after rotary evaporation under reduced pressure. The yield was 15%.

[0044] (2) Characterization of probes

[0045] 10-(2-Hydroxyphenyl)-5,15-(pentafluorophenyl)corrole (1): 1 H NMR (400 MHz, CDCl 3) δ 9.08 (d, J = 4.3 Hz, 2H, pyrrole-H), 8.74 (d, J = 4.7 Hz, 2H, pyrrole-H), 8.67 (d, J = 4.7 Hz, 2H, pyrrole-H), 8.58 (d, J = 4.2 Hz, 2H, pyrrole-H), 7.90 (d, J = 6.3 Hz, 1H, Ph-H), 7.68 (t, J = 7.9 Hz, 1H, Ph-H), 7.31 ((t,, J = 7.4 Hz, 2H, Ph-H), 7.27 (s, 1H, Ph-H). 19 F NMR (471 MHz, CDCl 3 ) δ -137.81 (ddd, J = 58.8, 24.1, 8.1 Hz, 4F), -152.44 (t, J = 20.8 Hz, 2F), -161.55 (td, J = 21.9, 21.1, 10.9 Hz, 4F). HRMS-ESI: m / z: calcd. for C 37 H 17 F 10 N 4 O: 723.1237, found: 723.1258.( Figure 1-3 )

[0046] 10-(3-Hydroxyphenyl)-5,15-(pentafluorophenyl)corrole (2): 1 H NMR (400 MHz, CDCl 3 ) δ 9.13 (d, J = 4.2 Hz, 2H, pyrrole-H), 8.69 (d, J = 4.8 Hz, 4H, pyrrole-H), 8.59 (d, J = 4.2 Hz, 2H, pyrrole-H), 7.70 (d, J = 7.4 Hz, 1H, Ph-H), 7.52 (t, J = 7.8 Hz, 1H, Ph-H), 7.35 (s, 1H, Ph-H), 6.99–6.93 (m, 1H, Ph-H). 19 F NMR (376 MHz, CDCl 3 ) δ -137.18– -138.30 (m, 4F), -152.80 (t, J = 20.9 Hz, 2F), -161.53– -162.16 (m, 4F). HRMS-ESI: m / z: calcd. for C 37 H 17 F 10 N 4 O: 723.1237, found: 723.1254.( Figure 4-6 )

[0047] 10-(4-Hydroxyphenyl)-5,15-(pentafluorophenyl)carbole (3): 1 H NMR (400 MHz, CDCl 3 )δ9.11(d,J=4.3Hz,2H,pyrrole-H),8.71(q,J=4.7Hz,4H,pyrrole-H),8.57(d,J=4.2Hz,2H,pyrrole-H),8.03(d,J=8.2Hz,2H,Ph-H),7.20(d,J=8.3Hz,2H,Ph-H). 19 F NMR (376 MHz, CDCl 3 )δ-137.88(dt,J=20.2,10.2Hz,4F),-152.90(t,J=21.0Hz,2F),-161.80(dt,J=23.7,7.9Hz,4F).HRMS-ESI:m / z:calcd.for C 37 H 17 F 10 N 4 O:723.1237,found:723.1257.( Figure 7-9 )

[0048] (2) Hydroxyl free corrole to Cu 2+ Fluorescent probe performance test

[0049] (1) Selectivity

[0050] ① Experimental methods

[0051] In a 3 mL cuvette, add 1 μM hydroxy-free corrole methanol solution, and then add 5 μM Cu(HCOO) 2 ·4H 2 O、CuSO 4 , CuCl 2 、FeCl 3 , KCl, NaCl, FeCl 2 ·4H 2 O、BaCl 2 ·2H 2 O、Mn(HCOO) 2 ·4H 2 O and AlCl 3 6H 2 O, mix well, scan the fluorescence emission spectrum. The maximum excitation wavelength of probe 1 is 420nm, the maximum emission wavelength is 636nm; the maximum excitation wavelength of probe 2 is 420nm, the maximum emission wavelength is 636nm; the maximum excitation wavelength of probe 3 is 418nm, the maximum emission wavelength is 636nm;

[0052] ② Experimental results

[0053] In the solutions of fluorescent probes 1, 2 and 3, fluorescence spectroscopy was used to detect common metal ions (such as Fe 3+ , K + 、Na + etc.) performed ion recognition. Figure 12-16 As shown in the figure, the fluorescence intensity of the hydroxyl free corrole probe is strong. 2+ After that, the fluorescence intensity decreased significantly, and fluorescence quenching occurred. + , K + , Fe 3+ , Fe 2+ , Mn 2+ and Ba 2+ In the presence of Al 3+ The fluorescence intensity of the probe decreased slightly in the presence of Cu. 2+ After adding the fluorescent probe solution, its bright red fluorescence turned colorless; while after adding other metal ions, the fluorescence color did not change, such as Figure 10-15 .

[0054] (2) Working curve and detection limit

[0055] ① Experimental methods

[0056] In a 3 mL cuvette, add 1 μM hydroxy-free corrole methanol solution, and then gradually add 5 mM Cu(HCOO) 2 ·4H 2 O solution, stir evenly and wait for 1 min, then detect the fluorescence intensity. 2 ·4H 2 O solution is added, the probe solution at this concentration changes the Cu 2+ After the titration, the corrole probe and the quantitative Cu 2+ The fluorescence spectrum of the probe after the reaction can be obtained by comparing its fluorescence intensity with that of Cu 2+ The relationship between the concentration changes is analyzed, and the linear relationship between the two is obtained by analyzing the spectrum.

[0057] ② Experimental results

[0058] Fluorescence titration experiments such as Figure 16-21 As shown. The 3 hydroxycorrole probe solutions showed bright red fluorescence, but when a trace amount of Cu 2+ After that, the fluorescence decreased significantly.2+ As the concentration increases, the fluorescence intensity of the hydroxycorrole probe gradually decreases until it is finally quenched. This may be due to the interaction of the hydroxycorrole probe with Cu 2+ A coordination reaction occurs to form a metal complex.

[0059] In addition, the hydroxycorrole probe and Cu 2+ The concentration of the three probes was linearly fitted with Cu 2+ There is a good linear relationship between the concentration of probe 1 and Cu 2+ There is an excellent linear relationship in the concentration range of 0-3.96μM, and the linear equation is F 636 =-1.96C Cu2+ +7.42, R 2 =0.9956, and the detection limit LOD is 31nM. For probe 2, its fluorescence intensity at 636nm is 2+ There is a good linear relationship with the increase of concentration, and the linear equation is F 636 =-1.63C Cu2+ +6.52, R 2 =0.9941 The standard deviation was calculated by testing the blank value of probe 2, and the detection limit was calculated to be 31 nM. For probe 3, its fluorescence intensity at 636 nm increased with the increase of Cu 2+ There is a linear decrease with the increase of concentration, and the linear equation is F 636 =-1.63C Cu2+ +5.08, R 2 =0.9996, and the detection limit LOD is 22nM.

[0060] The above is a preferred embodiment of the present invention. It should be pointed out that a person skilled in the art can make several improvements and modifications without departing from the principle of the present invention. These improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A fluorescent probe based on hydroxyl free corrole, characterized in that: The fluorescent probe based on hydroxyl free corrole is used to selectively identify copper ions. The structural formulas of the fluorescent probe based on hydroxyl free corrole are as follows:

2. A method for preparing a fluorescent probe based on hydroxyl free corrole as claimed in claim 1, characterized in that: The reaction formula of the method is:

3. The preparation method according to claim 2, characterized in that: The method comprises the following steps: weighing pentafluorophenyl dipyrrolidine and placing it in a round-bottom flask, adding salicylaldehyde or m-hydroxybenzaldehyde or p-hydroxybenzaldehyde, and dissolving it in dichloromethane (DCM); after stirring evenly, adding trifluoroacetic acid (TFA), reacting at room temperature for a period of time, adding triethylamine (Et3N), and adding an appropriate amount of dichloromethane after the smoke disappears; then adding 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ), stirring and reacting at room temperature for a period of time; separating by chromatography on a 100-200 mesh coarse silica gel column, using dichloromethane as an eluent, collecting a substance with red fluorescence under irradiation of a 365nm ultraviolet lamp, and performing reduced pressure rotary evaporation to obtain a crude product; separating the crude product by chromatography on a 300-400 mesh fine silica gel column, using dichloromethane and n-hexane as eluents, collecting a red fluorescent substance, and performing reduced pressure rotary evaporation to obtain a purple solid.

4. The preparation method according to claim 3, characterized in that: Methyl chloride and n-hexane were used as eluents in a ratio of 1:

1.

5. The preparation method according to claim 3, characterized in that: The method comprises the following steps: weighing 1.2 g of pentafluorophenyl dipyrrolidine and placing it in a 250 mL round-bottom flask, adding 135 μL of salicylaldehyde, and dissolving it in 60 mL of dichloromethane (DCM); after stirring evenly, adding 12 μL of trifluoroacetic acid (TFA), and reacting at room temperature for 4.5 hours; adding 12 μL of triethylamine (Et3N), and after the smoke disappears, adding 40 mL of dichloromethane; adding 0.8 g of 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ), stirring and reacting at room temperature for 1 hour; using a 100-200 mesh coarse silica gel column for chromatographic separation, using dichloromethane as an eluent, collecting a substance with red fluorescence under irradiation of a 365 nm ultraviolet lamp, and performing reduced pressure rotary evaporation to obtain a crude product; using a 300-400 mesh fine silica gel column for chromatographic separation of the crude product, using dichloromethane and n-hexane as eluents in a ratio of 1:1, collecting a red fluorescent substance, and performing reduced pressure rotary evaporation to obtain a purple solid.

6. The preparation method according to claim 3, characterized in that: The method comprises the following steps: weighing 1.2 g of pentafluorophenyl dipyrrolidine and placing it in a 250 mL round-bottom flask, adding 0.1655 g of m-hydroxybenzaldehyde, and dissolving it in 60 mL of dichloromethane (DCM); after stirring evenly, adding 12 μL of trifluoroacetic acid (TFA), and reacting at room temperature for 4.5 hours; adding 12 μL of triethylamine (Et3N), and after the smoke disappears, adding 40 mL of dichloromethane; adding 0.8 g of 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ), stirring and reacting at room temperature for 1 hour; using a 100-200 mesh coarse silica gel column for chromatographic separation, using dichloromethane as an eluent, collecting a substance with red fluorescence under irradiation of a 365 nm ultraviolet lamp, and performing reduced pressure rotary evaporation to obtain a crude product; using a 300-400 mesh fine silica gel column for chromatographic separation of the crude product, using dichloromethane and n-hexane as eluents in a ratio of 1:1, collecting a red fluorescent substance, and performing reduced pressure rotary evaporation to obtain a purple solid.

7. The preparation method according to claim 3, characterized in that: The method comprises the following steps: weighing 1.2 g of pentafluorophenyl dipyrrolidine and placing it in a 250 mL round-bottom flask, adding 0.1655 g of p-hydroxybenzaldehyde, and dissolving it in 60 mL of dichloromethane (DCM); after stirring evenly, adding 12 μL of trifluoroacetic acid (TFA), and reacting at room temperature for 4.5 hours; adding 12 μL of triethylamine (Et3N), and after the smoke disappears, adding 40 mL of dichloromethane; adding 0.8 g of 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ), stirring and reacting at room temperature for 1 hour; using a 100-200 mesh coarse silica gel column for chromatographic separation, using dichloromethane as an eluent, collecting a substance with red fluorescence under irradiation of a 365 nm ultraviolet lamp, and performing reduced pressure rotary evaporation to obtain a crude product; using a 300-400 mesh fine silica gel column for chromatographic separation of the crude product, using dichloromethane and n-hexane as eluents in a ratio of 1:1, collecting a red fluorescent substance, and performing reduced pressure rotary evaporation to obtain a purple solid.

8. Use of the hydroxyl free corrole based fluorescent probe according to claim 1 or the hydroxyl free corrole fluorescent probe prepared by the method according to any one of claims 2 to 7 in detecting copper ions.

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