CN114573571A

CN114573571A - Carbazole derivative fluorescent probe containing dicyano isophorone base and preparation method and application thereof

Info

Publication number: CN114573571A
Application number: CN202210250858.8A
Authority: CN
Inventors: 朱维菊; 杨逸娴; 刘丽; 方敏; 李村; 吴振玉
Original assignee: Anhui University
Current assignee: Anhui University
Priority date: 2022-03-15
Filing date: 2022-03-15
Publication date: 2022-06-03
Anticipated expiration: 2042-03-15
Also published as: CN114573571B

Abstract

The invention discloses a carbazole derivative fluorescent probe containing dicyano isophorone base, a preparation method and application thereof, wherein the structural formula of the carbazole derivative fluorescent probe containing dicyano isophorone base is as follows:

the carbazole derivative fluorescent probe has multiple functions, and can realize ClO through an ultraviolet-visible spectrophotometry method and a ratio fluorescence method^‑The visual identification and quantitative detection of the ions have good anti-interference performance, high selectivity and sensitivity under the condition of the existence of other ions.

Description

Carbazole derivative fluorescent probe containing dicyano isophorone base and preparation method and application thereof

Technical Field

The invention relates toCarbazole derivative fluorescent probe containing dicyanoisonophorone group, preparation method and application thereof, and particularly used for detecting ClO^-Ions, belonging to the field of ion detection and fluorescent molecular probes.

Background

Reactive Oxygen Species (ROS), which are byproducts of oxygen metabolism, have been shown to be important intracellular signaling molecules that are intimately involved in many fundamental biological processes and the pathogenesis of various diseases. ClO^-As one of ROS, it has a very strong bactericidal effect in the innate immune system. Under physiological conditions, ClO^-Can react with various biomolecules (such as amino acid, protein, membrane lipid, and DNA) rapidly, so that ClO with proper concentration^-Is beneficial and abnormal ClO^-Is harmful to human body, and can cause oxidative damage, and induce various human diseases, such as lung injury, rheumatoid arthritis, neuronal degeneration, cardiovascular diseases and cancer. At the same time, ClO^-Has double functions of bleaching and sterilizing, and is widely applied to daily life such as household bleaching, drinking water disinfection and the like. In view of the above-mentioned physiological and pathological properties of HClO, there is an urgent need for a specific ultra-rapid detection tool for visually detecting ClO in living cells^-The concentration of (2) is varied. Therefore, a method for rapidly, specifically and visually identifying ClO is developed^-Fluorescent probe of ions for real-time monitoring of ClO in biological cells or in water^-Ions are of great importance.

Disclosure of Invention

The invention aims to provide a carbazole derivative fluorescent probe containing dicyano isophorone base and a preparation method and application thereof, and aims to solve the technical problem that ClO can be detected and identified through molecular design synthesis^-The fluorescent probe of (1).

The carbazole derivative fluorescent probe containing dicyano isophorone base is a ratiometric fluorescent probe, and can still react with ClO in the presence of other ions^-The ions have higher selectivity and sensitivity, and the fluorescent probe and the ClO have higher sensitivity^-The obvious color change phenomenon generated by ion mixing can realize naked eye identification and colorimetric analysis.

The structural formula of the carbazole derivative fluorescent probe containing dicyano isophorone base is shown as follows:

the carbazole derivative containing dicyano isophorone-thiophene aldehyde group has good intramolecular charge transfer effect, C ═ C bonds contained in dicyano isophorone groups in molecules are often used as reaction sites of HClO, and HClO can oxidize the C ═ C bonds into aldehyde or ketone under the condition of normal temperature, so that the intramolecular charge transfer effect of the whole molecular system is changed, the ultraviolet and fluorescence phenomena of the original probe compound are changed, and the probe molecule can realize the change of the ClO^-And (4) rapidly detecting ions.

The invention relates to a preparation method of a carbazole derivative fluorescent probe containing dicyano isophorone base, which comprises the following steps:

step 1: synthesis of intermediate I-DCM

3-formyl-6-iodo-N-butylcarbazole (0.7430g,1.91mmol) and DCM (0.4051g,2.17mmol) were weighed out and dissolved in 25mL of ethanol, 0.6mL of piperidine was added dropwise to the round-bottom flask, and the reaction was stirred under reflux for 6 hours; after the reaction was complete, the product was precipitated from hot ethanol, filtered while hot, and the filter cake was washed three times with ethanol to give 0.7112g of a red solid.

And 2, step: synthesis of SFQ

150mg of I-DCM (0.27mg), 62.4mg of 5-formyl-2-thiopheneboronic acid (0.40mg) and 31mg of Pd (PPh) were taken respectively₃)₄(0.27mmol) was poured into a three-necked flask and charged with N₂Ensuring oxygen-free environment, injecting 20mL of mixed solution of toluene and ethanol (3:1, V/V) and 3mL of saturated K₂CO₃Solution, the reaction is carried out at 80 ℃ for 24 hours; after the reaction was complete, the reaction was cooled to room temperature, the product was poured into a large amount of distilled water, extracted with dichloromethane and the organic phases were combined (three extractions on average), and the organic phase was extracted with anhydrous MgSO₄Drying overnight, filtering, and then carrying out reduced pressure spin-drying on the filtrate to obtain a crude product; using petroleum ether, dichloromethane and acetic acidEthyl ester (8:1:1, V/V/V) was used as eluent, and purified by silica gel column chromatography to obtain 54mg of red solid.

The synthetic route of the invention is as follows:

the invention relates to an application of a carbazole derivative fluorescent probe containing dicyano isophorone base in qualitative or quantitative detection of ClO^-When used, the reagent is used as a detection reagent.

Further, ultraviolet-visible absorption spectrometry is carried out in an organic solvent medium, and ClO is realized through the change of the solution color^-Qualitative or quantitative detection of (a).

Further, fluorescence spectrometry is carried out in an organic solvent medium, and ClO is realized through the change of dual-channel fluorescence intensity^-Qualitative or quantitative detection of (a).

The organic solvent medium is DMF.

The fluorescent probe can be used for ClO^-The identification and detection of the ions have stronger anti-interference capability on various other ions, and the fluorescent probe and the ClO of the invention^-The obvious color change phenomenon generated by ion mixing can realize naked eye identification and colorimetric analysis.

The invention has the beneficial effects that:

the fluorescent probe compound has multiple functions, and can realize ClO respectively by ultraviolet-visible spectrophotometry and fluorescence spectrometry^-And (4) identifying ions. The fluorescent probe can specifically identify ClO^-Ion, adding ClO into DMF solution of probe^-Ion, a visible absorption band at 468nm that is significantly red-shifted by 25nm in the ultraviolet spectrum with a concomitant decrease in the absorption intensity; the probe solution showed orange color under normal room light, and ClO was added^-After ionization, the solution changed to pink, and under a 365nm ultraviolet lamp, the solution changed from orange-red to cyan. In the fluorescence spectrum, ClO^-Addition of ions toThe fluorescence at 473nm of the probe solution increases, the emission position gradually red-shifts to 486nm, at the same time, the fluorescence at 613nm decreases and the emission peak disappears, showing a ratiometric fluorescence phenomenon, and the probe SFQ shows two different emission channels, which enables it to detect ClO-ions in a ratiometric manner in a very short time. The probe can realize the ClO reaction by ultraviolet-visible spectrophotometry and fluorescence spectrometry^-The ion identification and quantitative detection still has good anti-interference performance, high selectivity and sensitivity under the condition of the existence of other ions.

The fluorescent probe can be used for ClO in real water samples^-The ions are rapidly identified and quantitatively detected, and the ClO is also detected^-The ion identification has higher selectivity and better anti-interference capability, and the obvious color change phenomenon can realize naked eye identification and colorimetric analysis. The real water sample test shows that the probe has good prospect in the application of the actual water sample; the research on the practicability of the probe in the aspect of biological application shows that the probe solution can be used for exogenous ClO in cells^-And detecting the ions. The experimental results show that the probe has good application potential in environmental monitoring and organisms.

Drawings

FIG. 1a shows the UV absorption spectra of a fluorescent probe (SFQ) of the present invention with different ions added to DMF (ClO is shown in the inset)^-Color change before and after).

FIG. 1b shows fluorescence spectra of fluorescent probes (SFQ) of the present invention with different ions added in DMF (ClO is shown in the inset)^-Color change before and after irradiation with 365nm uv lamp).

FIG. 2a shows the fluorescent probe (SFQ) (10. mu.M) of the present invention added with ClO at different concentrations in DMF^-Ultraviolet absorption titration spectrum (0-32.5. mu.M).

FIG. 2b shows the addition of different concentrations of ClO to a fluorescent probe (SFQ) (10. mu.M) according to the invention in pure DMF^-(0-40. mu.M) fluorescence titration spectrum at 370nm excitation.

FIG. 2c shows the addition of ClO at different concentrations to a fluorescent probe (SFQ) (10. mu.M) according to the invention in pure DMF^-(0-40. mu.M) fluorescence titration spectra at 470nm excitation.

FIG. 3a shows the UV absorption intensity of fluorescent probe (SFQ) (10. mu.M) of the present invention and different concentrations of ClO^-(12.5-25. mu.M).

FIG. 3b shows the fluorescence intensity ratio F of the fluorescent probe (SFQ) of the present invention₄₈₉/F₆₁₃And ClO^-Concentration (13-27.5. mu.M).

FIG. 4a shows fluorescent probes (SFQ) (10. mu.M) of the invention in ClO^-Ultraviolet absorption response in the presence of added other analytes.

Uv absorption of SFQ after addition of other analytes (2 equiv.);

UV absorption of SFQ in the presence of ClO-and other analytes (2 equiv.).

FIG. 4b shows the fluorescent probe SFQ (10. mu.M) in ClO^-Fluorescence intensity ratio in the Presence of addition of other analytes (F)₄₈₉/F₆₁₃)。

Represents the fluorescence intensity ratio (F) of SFQ after addition of other analytes (2equiv.)₄₈₉/F₆₁₃)；

Represents the fluorescence intensity ratio (F) of SFQ in the presence of ClO-and other analytes (2equiv.)₄₈₉/F₆₁₃)。

FIG. 5 shows that ClO is added to the fluorescent probe SFQ at different concentrations^-Later change in fluorescence intensity at 489nm with time (lambda)_ex＝370nm)。

FIG. 6 shows addition of ClO to SFQ as a fluorescent probe of the present invention^-Mass spectrum of the post product.

FIG. 7 shows fluorescent probes SFQ (10. mu.M) and ClO of the present invention^-(3.0equiv.) in DMSO-d₆Nuclear magnetic titration hydrogen spectrogram in (1).

FIG. 8 is a graph comparing the fluorescence intensity captured in HepG2 cells.

Detailed Description

The invention is further illustrated by, but is not limited to, the following examples.

Example 1: synthesis of fluorescent Probe SFQ

3-formyl-6-iodo-N-butylcarbazole (0.7430g,1.91mmol) and DCM (0.4051g,2.17mmol) were weighed out and dissolved in 25mL of ethanol, 0.6mL of piperidine was added dropwise to the round-bottom flask, and the reaction was stirred under reflux for 6 hours. At the end of the reaction, the product was precipitated from hot ethanol, filtered while hot and the filter cake was washed three times with ethanol to give 0.7112g of a red solid (I-DCM). The yield was 68%.

¹H NMR(600MHz,DMSO-d₆)δ8.68(s,1H),8.57(s,1H),7.82(d,J＝8.6Hz,1H),7.75(d, J＝8.6Hz,1H),7.67(d,J＝8.6Hz,1H),7.53(d,J＝8.6Hz,1H),7.47(d,J＝8.4Hz,2H),6.85(s, 1H),4.40(t,2H),2.64(s,2H),2.61(s,2H),2.51(s,2H),1.74(m,2H),1.29(m,2H),1.06(s,6H), 0.88(t,3H).[M-H]⁺:545.1249.

150mg of I-DCM (0.27mg), 62.4mg of 5-formyl-2-thiopheneboronic acid (0.40mg) and 31mg of Pd (PPh) were taken respectively₃)₄(0.27mmol) was poured into a three-necked flask and charged with N₂Ensuring oxygen-free environment, injecting 20mL of mixed solution of toluene and ethanol (3:1, V/V) and 3mL of saturated K₂CO₃The solution was reacted at 80 ℃ for 24 hours. After the reaction was complete, the reaction was cooled to room temperature, the product was poured into a large amount of distilled water, extracted with dichloromethane and the organic phases were combined (three extractions on average), and the organic phase was extracted with anhydrous MgSO₄Drying overnight, filtering, and spin-drying the filtrate under reduced pressure to obtain the crude product. Then, it was purified by silica gel column chromatography using petroleum ether, dichloromethane and ethyl acetate (8:1:1, V/V/V) as eluent to obtain 54mg of red Solid (SFQ) with a yield of 37.5%.

¹H NMR(400MHz,DMSO-d₆)δ9.87(s,1H),8.71(s,1H),8.65(s,1H),8.04(d,J＝3.9Hz, 1H),7.90(d,J＝8.5Hz,1H),7.80(d,J＝9.7Hz,1H),7.78–7.62(m,3H),7.50–7.39(m,2H), 6.84(s,1H),4.42(t,2H),2.59(d,J＝6.8Hz,4H),1.79–1.68(m,2H),1.30–1.24(m,2H),1.02(s, 6H),0.83(t,3H).¹³C NMR(101MHz,CDCl₃)δ182.69(s),169.37(s),155.74(s),154.59(s), 142.04(s),141.61(d,J＝3.2Hz),138.19(s),137.93(s),127.68(s),127.01(s),126.10(s),125.08 (d,J＝3.6Hz),123.52-123.10(m,3C),122.72(s),120.74(s),118.65(s),113.90(s),113.19(s), 109.90(d,J＝2.9Hz),43.41(s),43.08(s),39.37(s),32.14(s),31.21(s),29.78(s),28.16(s),20.60 (s),13.92(s).IR(KBr,cm^-1)2210(-CN),1384(-CH₃),674(C-S).[M-H]⁺:529.2154.

Example 2: selective spectroscopic study of fluorescent probes SFQ

The selectivity is one of the parameters of the probe for detecting the ions, and has great significance. As can be seen from FIG. 1a, the probe SFQ (10. mu.M) showed a strong absorption peak at 468nm in N, N-Dimethylformamide (DMF) solution, which is probably due to N-. pi.within SFQ^*And (4) transition. Each of the analytes (Cl) was added to the probe SFQ in an amount of 2 times equivalent to each other^-，HSO₃ ^-， SO₃ ^2-，HCO₃ ^-，SCN^-，CH₃COO^-，ONOO^-，NO₂ ^-，H₂O₂，Cu²⁺，Fe³⁺，Na⁺，Mg²⁺，Co²⁺Cys, GSH and Lysine), its UV absorption is essentially unchanged, only ClO is present^-The absorption band of SFQ at 468nm was clearly red-shifted by 25nm with a concomitant decrease in the absorption intensity (FIG. 1 a). The probe SFQ solution shows orange under normal room light, and ClO is added^-The solution then turns pink in color (inset in fig. 1a), a phenomenon that coincides with a change in uv absorption. In the fluorescence spectrum, ClO^-The addition of (2) causes the SFQ solution to exhibit a ratiometric fluorescence phenomenon, in which the fluorescence at 473nm increases by nearly 10-fold, the luminescence position is red-shifted to 486nm, and at the same time, the fluorescence at 613nm decreases and the emission peak disappears (FIG. 1 b). Under a 365nm UV lamp, the solution changed color from orange-red to cyan (inset in FIG. 1b), while the addition of the other analytes did not change significantly. The above results indicate that the probe SFQ is only for ClO^-Has high selectivity.

Example 3: titration Spectroscopy study of fluorescent probes (SFQ)

Probes SFQ vs ClO^-The UV and fluorescence titration spectroscopy experiments were further explored. As shown in fig. 2a, with ClO^-The absorption peak of SFQ at 469nm gradually red-shifted and slowly decayed while the peak at 364nm gradually shifted to 370 nm. Probes SFQ (10. mu.M) and ClO^-Has a good linear relationship (R) in the range of 12.5-25 μ M²＝0.9919)，A₄₆₉＝-0.0064[ClO^-]+0.2558 (fig. 3 a). According to DL being 3 sigma/K, the probe SFQ is calculated to be opposite to ClO in the ultraviolet-visible absorption spectrum^-The detection limit of (2) is 1.13. mu.M. FIGS. 2b and 2c show the addition of ClO at different concentrations to the probe SFQ (10. mu.M), respectively^-(0-40. mu.M) fluorescence titration spectra at 470nm and 370nm excitation. As shown in FIG. 2b, at 470nm excitation, the emission of the probe at 613nm gradually decreased, while at 370nm excitation, the emission of the probe at 473nm gradually increased and the emission peak position gradually shifted to 489 nm. To this end, we make the fluorescence intensity ratio F₄₈₉/F₆₁₃And ClO^-Concentration as a function of fluorescence intensity ratio F in the range of 13-27.5. mu.M₄₈₉/F₆₁₃And ClO^-The concentration shows a good linear relation (R)²＝0.9930)，F₄₈₉/F₆₁₃＝1.5126[ClO^-]-12.1015 (fig. 3 b). According to DL being 3 sigma/K, the probe SFQ is calculated to be corresponding to ClO in the fluorescence spectrum^-The detection limit of the ratio of (A) to (B) is 0.14. mu.M.

Example 4: competitive study of fluorescent probes (SFQ)

To explore the probe SFQ pair ClO^-For the detection of interference resistance, a competitive experiment of an ultraviolet-visible absorption spectrum and a fluorescence spectrum is carried out. In the ultraviolet absorption spectrum, it was shown that only in ClO^-The ultraviolet absorption of the probe SFQ is reduced in the existing system (FIG. 4a), and the ClO is hardly influenced by the existence of other analytes^-Detection of (3). Similarly, only in ClO^-Fluorescence intensity ratio (F) of Probe SFQ in the Presence of System₄₈₉/F₆₁₃) Ming dynastyIncrease by dozens of times, the presence of other analytes is hardly possible to ClO^-Caused interference with the detection of (2) (FIG. 4b), probe SFQ on ClO^-Has high selectivity and interference resistance. Therefore, the probe is expected to become a sensor which can be developed and applied to finish the ClO in a complex environment^-Detection of (3).

Example 5: time response of fluorescent probes (SFQ)

As is well known, the rapid time response is of great importance to the detection of various analytes such as anions, cations and amino acids by a fluorescent probe in practical application. As shown in FIG. 5, 12.5. mu.M, 25. mu.M and 50. mu.M are respectively added into the probe SFQ (10. mu.M, DMF), the response is fast within 5 seconds, the equilibrium is basically reached within 20 seconds, and the real-time detection of ClO as a transient metabolite is satisfied^-The requirements of (1). We consult the last few years of ClO detection at home and abroad^-The probe pair ClO can be seen by comparing the related documents^-The response of (A) is obviously faster than that of the probes reported in other literatures.

Example 6: fluorescent probes SFQ vs ClO^-Research on response mechanism

To study the probes SFQ vs ClO^-The response mechanism of (1) we put probes SFQ to ClO^-The product after reaction was subjected to mass spectrometry and nuclear magnetic titration. As shown in FIG. 6, one peak m/z-481.2607 can be attributed to [ SFQ-O + H]⁺This may be that-C ═ C (cn) is oxidized to-C ═ O. To continue to verify this guess, we performed a nuclear magnetic titration test, as can be seen from the experimental results: ha, Hb, Hc, and Hd all shift to high fields with chemical shifts from δ 7.46ppm, 7.43ppm, 6.84ppm, 8.65ppm to 6.50ppm, 6.42ppm, 5.69ppm, and 8.52ppm (fig. 7), respectively, probably due to oxidation of two strong electron-withdrawing groups (-CN) to a medium electron-withdrawing group (-C ═ O). It was thus confirmed that the electron-withdrawing CN group of the probe SFQ was exposed to ClO^-Thereby being oxidized into a keto group.

Example 7: fluorescent probe SFQ for ClO in water sample^-Detection of (2)

To examine the potential application of SFQ in real life, lake water and fruit around Anhui university were selectedLaboratory tap water for example for real water sample internal test ClO^-. DMF was used to prepare a solution of probe SFQ (10. mu.M), and lake water and tap water were used to prepare 10 solutions^-2ClO of M^-And (3) solution. Adding three ClO solutions of 5. mu.M, 10. mu.M, 15. mu.M and 20. mu.M, respectively^-The fluorescence intensity of the solution was measured. As can be seen from Table 1, ClO was measured^-The concentration and the added concentration have small error, the test recovery rate is in the range of 98.5-103 percent, and the result shows that the probe SFQ detects the ClO in a real water sample^-Has very high accuracy, and can be applied to ClO in real water samples^-Detection of (3).

TABLE 1 fluorescent Probe SFQ of the invention for ClO in various real water samples^-Detection of (3).

Example 8: fluorescent probe compounds SFQ vs ClO in cells^-Fluorescent imaging test of

By confocal microscopy, HepG2 cells stained for 30 min with SFQ (10. mu.M) were observed, capturing fluorescence in both the red and cyan channels, as ClO was added to the cells^-After 0.5 hour incubation (50 μ M), a significant decrease in fluorescence was observed in the red channel, while a stronger fluorescence emission was exhibited in the cyan channel. The intensity of fluorescence captured in the cells is shown in FIG. 8, and the confocal imaging of the cells is consistent with the experimental phenomenon. Therefore, the probe SFQ has stronger cell membrane penetrating capability and can detect the endogenous and exogenous ClO in the cells through the change of dual-channel fluorescence^-Distribution of (2).

Claims

1. A carbazole derivative fluorescent probe containing dicyano isophorone base is characterized in that the structural formula is as follows:

2. the method for preparing the dicyanoisoprophorone group-containing carbazole derivative fluorescent probe according to claim 1, characterized by comprising the steps of:

step 1: synthesis of intermediate I-DCM

Weighing 3-formyl-6-iodine-N-butylcarbazole and DCM, dissolving in ethanol, dropwise adding piperidine into a round-bottom flask, and stirring for reaction under a reflux state; after the reaction is finished, precipitating a product from hot ethanol, filtering while the product is hot, and washing a filter cake with ethanol to obtain red solid I-DCM;

step 2: synthesis of SFQ

Respectively taking I-DCM, 5-aldehyde-2-thiopheneboronic acid and Pd (PPh)₃)₄Adding into a reactor, charging N₂Ensuring an oxygen-free environment, injecting a mixed solution of toluene and ethanol and saturated K₂CO₃Solution, reacting at 80 ℃; after the reaction is finished, cooling to room temperature, separating and purifying to obtain a target product which is a red solid;

the reaction scheme is as follows:

3. use of the dicyanoisoprophorone group-containing carbazole derivative fluorescent probe according to claim 1, characterized in that: the carbazole derivative fluorescent probe containing dicyano isophorone base is used for qualitatively or quantitatively detecting ClO^-When used, the reagent is used as a detection reagent.

4. Use according to claim 3, characterized in that:

performing ultraviolet-visible absorption spectrum measurement in an organic solvent medium, and realizing ClO through the change of solution color^-Qualitative or quantitative detection of (a).

5. Use according to claim 4, characterized in that:

the organic solvent medium is DMF.

6. Use according to claim 3, characterized in that:

fluorescence spectrum measurement is carried out in an organic solvent medium, and ClO is realized through the change of dual-channel fluorescence intensity^-Qualitative or quantitative detection of (a).

7. Use according to claim 6, characterized in that:

the organic solvent medium is DMF.

8. Use according to claim 4 or 6, characterized in that:

the carbazole derivative fluorescent probe containing dicyano isophorone base is used for detecting ClO in ultraviolet-visible absorption spectrum^-Has a detection limit of 1.13 mu M for ClO in a fluorescence spectrum^-The detection limit of the ratio of (A) to (B) is 0.14. mu.M.