Long-wavelength H2S fluorescent probe as well as preparation method and application thereof

Abstract

The invention provides a long-wavelength H2S fluorescent probe as well as a preparation method and application thereof. The structural formula of the fluorescent probe is as follows: # imgabs0 #. The long-wavelength H2S fluorescent probe disclosed by the invention is simple in synthesis method and good in water solubility, and a product is easy to separate and purify. The fluorescent probe can identify HS <-> ions in an actual water sample, the fluorescent probe can identify H2S through long-wavelength (640nm) fluorescence enhancement in a water medium, the selectivity is high, the sensitivity is good, the detection limit reaches 10 <-6 > mol / L, and the fluorescent probe can be applied to detection of HS <-> in the actual water sample and living cells.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical synthesis analysis and detection, and specifically relates to a long-wavelength H2S fluorescent probe and a preparation method and application thereof. Background Art

[0002] As we all know, hydrogen sulfide (H2S) is a toxic gas with a rotten egg smell. It plays a key role in biological systems and is recognized as the third endogenous gas signal molecule besides nitric oxide (NO) and carbon monoxide (CO). In mammalian cells, it is produced by the action of enzymes such as cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE), and 3-mercaptopyruvate sulfotransferase (3-MST). Endogenous hydrogen sulfide has harmful effects on the biological cardiovascular system, such as vasodilation, inhibition of vascular smooth muscle cell proliferation, and negative myocardial inotropic effects. The content of hydrogen sulfide is also closely related to diseases such as Alzheimer's disease, Down syndrome, and cirrhosis. In addition, hydrogen sulfide can act as an antioxidant in the body to remove a variety of oxidative substances. Therefore, the detection of hydrogen sulfide in the biological environment has important prospects.

[0003] In recent years, there have been many designs based on the recognition of H2S fluorescent probe molecules, for example, ChemComm (2017), 53, 8759-8762; Sensors and Actuators, B: Chemical (2018), 255 (Part_2), 2347-2355; Journal of Materials Chemistry B: Materials for Biology and Medicine (2017), 5 (11), 2172-2180; Chinese Chemical Letters (2017), 28 (2), 218-221; J. Mater. Chem. B, (2017), 5, 2172-2180; Analyst, (2014), 139, 1945-1952. Although these documents can recognize H2S, the preparation process is long and the operation is complicated. Chinese Journal of Inorganic Chemistry(2013),11,2283-2288;RSC Adv.,(2016),6,85529-85537;Chem. Commun.,(2016),52,3131-3134;Dalton Trans.,(2017),46,12856-12864;Dalton Trans.,(2017),46,12856-12864;Analyst,(2014),139,1945-1952;J.Mater.Chem.B,(2018),Advance Article;Chem. Commun.,(2017),53,4791-4794;Although these documents can specifically identify H2S, their emission wavelength is short and long-wave identification cannot be achieved, and most of them cannot be identified in water. The reported fluorescent probes have certain limitations, while the long-wavelength emission probes have the advantages of strong anti-interference ability, little environmental influence, good water solubility, stable fluorescence imaging, high sensitivity, etc. Due to the urgent need to detect H2S at long wavelengths, it is of great significance to design and synthesize simpler and superior H2S fluorescent probes. Summary of the invention

[0004] The purpose of the present invention is to provide a long-wavelength H2S fluorescent probe and its preparation method and application to solve the technical problems existing in the background technology. The fluorescent probe has the advantages of simple synthesis route, long-wavelength emission, can recognize H2S in actual water samples and living cells, has specific selectivity, simple synthesis, etc.

[0005] The technical solution adopted by the present invention is as follows:

[0006] A long-wavelength H2S fluorescent probe, the structural formula of which is as follows:

[0007]

[0008] Based on the same technical idea, the present invention provides a method for preparing the above-mentioned long-wavelength H2S fluorescent probe, comprising the following steps:

[0009] (1) dissolving 3-hydroxy-3-methyl-2-butanone and malononitrile in ethanol, then adding dry ethanol to heat and reflux, cooling after the reaction is completed, filtering the precipitated solid, washing the solid with cold ethanol, and then dissolving the washed solid in ethanol for recrystallization, cooling after completion, filtering the precipitated solid, and obtaining pure compound 2;

[0010] (2) dissolving the obtained compound 2 and 4-diethylamino salicylaldehyde in ethanol, heating again under alkaline conditions for reflux reaction, and after the reaction is completed, drying the obtained crude product and purifying it by thin layer column chromatography to obtain compound 1;

[0011] (3) Compound 1, 2,4-dinitrofluorobenzene and anhydrous potassium carbonate are dissolved in a polar aprotic solvent and reacted by stirring at room temperature. After the reaction is completed, the solid is filtered out and then spin-dried to obtain a crude product. The crude product is purified by thin layer column chromatography to obtain a H2S fluorescent probe.

[0012] Further preferably, in the scheme of the present invention: sodium ethoxide is also added as a catalyst in the reflux reaction in step (1), and the mass ratio of 3-hydroxy-3-methyl-2-butanone to malononitrile to sodium ethoxide is (8-12):(10-15):1.

[0013] Further preferably, in the scheme of the present invention: the temperature of the heating reflux reaction in step (1) is 120°C to 150°C.

[0014] Further preferably, in the scheme of the present invention: the thin layer column chromatography purification in step (2) and step (3) is performed using ethyl acetate and petroleum ether as eluents for separation; the volume ratio of ethyl acetate to petroleum ether in step (2) is 1:5, and the volume ratio of ethyl acetate to petroleum ether in step (3) is 1:3.

[0015] Further preferably, in the scheme of the present invention: in the step (2), the mass ratio of compound 2 to 4-diethylaminosalicylaldehyde is 5:(3-4).

[0016] Further preferably, in the scheme of the present invention: in the step (3), the mass ratio of compound 1 to 2,4-dinitrofluorobenzene and anhydrous potassium carbonate is (1-2):(1-1.5):1.

[0017] Further preferably, in the scheme of the present invention: the polar aprotic solvent in step (3) is N,N-dimethylformamide.

[0018] Further preferably, in the scheme of the present invention: in the step (3), the reaction is stirred at room temperature for 8 to 24 hours.

[0019] Based on the same technical idea, the present invention provides an application of a long-wavelength H2S fluorescent probe, which detects H2S in a buffer solution of DMF and PBS with a pH of 7-11 and a volume ratio of 3:7.

[0020] Based on the same technical idea, the present invention provides an application of a long-wavelength H2S fluorescent probe, which detects H2S in actual water samples.

[0021] Based on the same technical idea, the present invention provides an application of a long-wavelength H2S fluorescent probe to detect HS in MCF-7 living cells. - Perform detection imaging.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The long-wavelength H2S fluorescent probe of the present invention has a simple synthesis method, good water solubility, and easy product separation and purification. It can identify HS in actual water samples. - Ions, fluorescent probes can identify H2S in water medium by long-wavelength (640nm) fluorescence enhancement, with high selectivity and good sensitivity, and the detection limit reaches 10 -6 mol / L, and can be applied to detect HS in actual water samples and living cells - .

[0024] (2) The present invention uses 4-diethylamino salicylaldehyde as a substrate, introduces a strong electron-withdrawing group compound 2 (tricyanofuran) by conjugation to construct a D-π-A structure, utilizes the intramolecular charge transfer (ICT) effect to lengthen the emission wavelength of the molecule and increase the Stokes shift, and introduces a recognition group (2,4-dinitrofluorobenzene) for specific recognition. A fluorescent (turn-on) probe L is designed and synthesized, and the synthetic route is simple, easy to prepare, and has a high yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is the fluorescent probe L of the present invention 1 H NMR spectrum;

[0026] Figure 2 is the fluorescent probe L of the present invention 13 C NMR spectrum;

[0027] Figure 3 is the mass spectrum of the fluorescent probe L of the present invention;

[0028] Figure 4 The fluorescent probe L and Br - , I - , NO2 - ,CO3 2- , HCO3 - , CH3COO - , HPO4 2- , H2PO4 - ,PO4 3- , SCN - , HS - , SO4 2- , SO3 2- , HSO3 - , HSO4 - , N3 - , S2O3 2- Fluorescence emission spectra before and after action;

[0029] Figure 5 The fluorescent probe L of the present invention is HS - Fluorescence detection diagram to resist interference from other metal ions during identification;

[0030] Figure 6 It is a graph showing the change of the fluorescence emission spectrum of the fluorescent probe L of the present invention before and after the action of different multiples of H2S;

[0031] Figure 7 is the detection limit diagram of the fluorescent probe L of the present invention;

[0032] Figure 8 This is the time response diagram of the fluorescent probe L of the present invention recognizing H2S;

[0033] Fig. 9 This is a test diagram of the effect of the response of the fluorescent probe L of the present invention to H2S in pH;

[0034] Fig.10 This is a graph showing the changes of the fluorescent probe L of the present invention after it acts on H2S of different concentrations in actual water samples;

[0035] Fig.11 This is an imaging diagram of the fluorescent probe L of the present invention in living cells. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is described and illustrated in conjunction with the following embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. Based on the embodiments provided by the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work belong to the scope of protection of the present invention. If not otherwise specified, all embodiments of the present invention and optional embodiments can be combined with each other to form new technical solutions.

[0037] Unless otherwise defined, all professional terms used below have the same meanings as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0038] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0039] The long-wavelength H2S fluorescent probe provided by the present invention is synthesized based on 4-diethylamino salicylaldehyde derivatives, and the synthesis route is as follows:

[0040]

[0041] There is no special limitation on the use of the fluorescent molecular probe of the present invention. Usually, the probe molecule can be dissolved in water containing 30% N,N-dimethylformamide and tested at room temperature. The detection principle of the fluorescent molecular probe of the present invention for H2S is shown in the following formula:

[0042]

[0043] When H2S is added, the fluorescence of the probe is enhanced because H2S can deprotect the hydroxyl group protected by 2,4-dinitrobenzene to generate a fluorescent compound molecule 1.

[0044] The specific synthesis method of the long-wavelength H2S fluorescent probe of the present invention is as follows:

[0045] (1) The specific synthesis steps of compound 2 are as follows:

[0046]

[0047] Compound 3-hydroxy-3-methyl-2-butanone (10.2 g), malononitrile (13.2 g) and sodium ethoxide (1.02 g) were dissolved in ethanol and stirred for 1 h. 15 ml of dry ethanol was added and heated to reflux for 1 h. The heating reaction temperature was 130°C, and then cooled in a refrigerator. The precipitated solid was filtered. The solid was first washed with cold ethanol, and then the washed solid was dissolved in ethanol for recrystallization. After completion, it was cooled, the precipitated solid was filtered to obtain a pure white solid. 17.48 g of compound 2 was isolated with a yield of 74.7%.

[0048] (2) The specific synthesis steps of compound 1 are as follows:

[0049]

[0050] Compound 2 (5.0 g), 4-diethylaminosalicylaldehyde (3.74 g), and NaOH (1.7 g) were dissolved in ethanol and refluxed for 12 h. The crude product was purified by thin layer column chromatography using EA:PE=1:5 (v / v) as eluent. After separation and spin drying, 2.44 g of compound 1 was obtained with a yield of 53.9%.

[0051] mp244.4-245.0℃; 1 H NMR (400MHz, DMSO-d6) δ10.87 (s, 1H), 8.23 ​​(d, J = 13.6

[0052] Hz,1H),7.71(d,J=9.2Hz,1H),6.95(d,J=13.6Hz,1H),6.46(dd,J=9.2,2.4Hz,1H) ,6.16(d,J=2.4Hz,1H),3.46(q,J=7.0Hz,4H),1.70(s,6H),1.16(t,J=7.0Hz,6H);

[0053] 13 C NMR (100MHz, DMSO-d6) δ177.98,175.71,162.69,154.51,114.55,113.66,113.31,112.43,107.22,97.66,96.86,49.15,45.00,26.37,13.11.

[0055] HRMS(ESI+)Calcd for C 22 H 22 N4NaO2[M+Na] + :397.1635,found:397.1218.

[0056] (3) The specific synthesis steps of the long-wavelength H2S fluorescent probe L are as follows:

[0057]

[0058] Compound 1 (374.44 mg), 2,4-dinitrofluorobenzene (223 mg), potassium carbonate (207 mg) were dissolved in DMF (15 mL) and stirred, and reacted at room temperature for 12 hours. After the reaction, the crude product was spin-dried to obtain a crude product, which was purified by thin layer column chromatography using EA:PE=1:3 (v / v) as the eluent to obtain 243.7 mg of probe L with a yield of 45.1%.

[0059] The H NMR spectrum of the synthesized H2S fluorescent probe L is shown in Figure 1 As shown, the NMR carbon spectrum of the synthesized H2S fluorescent probe L is as follows Figure 2 As shown, the mass spectrum of the synthesized H2S fluorescent probe L is shown in Figure 3 shown.

[0060] The structural characterization data of the synthesized H2S fluorescent probe L are as follows:

[0061] mp251.3-252.7℃; 1 H NMR (400MHz, DMSO-d6) δ8.92 (d, J=2.4Hz, 1H), 8.46

[0062] (dd,J=9.3,2.4Hz,1H),8.12(d,J=9.3Hz,1H),7.99(d,J=15.9Hz,1H),7.14(d,J=9.3Hz,1H),6.97(d,J=15.9H z,1H),6.87(d,J=9.3Hz,1H),6.69(d,J=2.1Hz,1H),3.49(q,J=6.8Hz,4H),1.63(s,6H),1.12(t,J=6.8Hz,6H);

[0063] 13 C NMR (101MHz, DMSO-d6) δ177.97,175.59,155.69,155.56,153.63,141.70,141.04,138.74,132.27,130.54,122. 59,118.64,113.76,113.34,112.85,112.51,111.49,110.22,103.90,98.78,92.79,52.04,44.96,25.54,12.94.

[0064] HRMS(ESI+)Calcd for C 28 H 24N6NaO6[M+Na] + :563.1650,found:563.1091.

[0065] Performance index test:

[0066] Selective detection of H2S by fluorescent probe L:

[0067] 2×10 -4 mol / L of various anions were added to the prepared 10 μmol / L probe L test solution DMF / PBS (3 / 7, v / v, pH=7.4), such as Figure 4 As shown. The probe L itself has weak fluorescence. When H2S is added, the fluorescence intensity of L at the emission wavelength of 640nm is significantly enhanced. After adding H2S, the fluorescence color of the probe L can be observed to change under the irradiation of the excitation wavelength of 543nm, that is, from no fluorescence to red fluorescence, which can be identified by the naked eye. After adding other anions, the fluorescence of L did not change significantly. The emission wavelength of probe L is 640nm, reaching the near-infrared, which can effectively minimize self-absorption and reduce the interference of autofluorescence, which is beneficial to biological imaging. In addition, HSO3 - May cause a slight decrease in fluorescence intensity, H2PO4 - The emission wavelength may be blue-shifted, while other anions do not cause significant changes. These results demonstrate that L has excellent selectivity for H2S recognition.

[0068] Anti-interference detection of H2S by fluorescent probe L:

[0069] 10 μmol / L fluorescent probe L in DMF:PBS=3:7 (v / v, pH=7.4) solution, and then other anions (2×10 -4 mol / L) after the fluorescence of L did not change significantly. Figure 5 As shown. Add 2×10 -4 mol / L HS - After that, except HSO3 - In addition, in various anions (Br - , I - , NO2 - ,CO3 2- , HCO3 - , CH3COO - , HPO4 2- , H2PO4 - ,PO4 3- , SCN - , HS - , SO4 2- , SO3 2- , HSO3 -, HSO4 - , N3 - , S2O3 2- ) still produces a significant fluorescence enhancement effect on the fluorescence of L, indicating that L has an important effect on HS - Can detect HS in the presence of other anions - , strong anti-interference ability.

[0070] Titration test of H2S by fluorescent probe L:

[0071] 10 μmol / L of fluorescent probe L in DMF:PBS=3:7 (v / v, pH=7.4) buffer solution, and 0-60×10 -6 mol / L HS - When HS - With the gradual addition of , the emission of probe L solution at 660 nm blue shifted to 640 nm, and the fluorescence intensity gradually increased with the increase of H2S concentration, as shown in Figure 6 When the H2S concentration is 60×10 -6 mol / L, the fluorescence intensity reached saturation.

[0072] Detection limit test of fluorescent probe L for H2S:

[0073] In the DMF:PBS=3:7 (v / v, pH=7.4) buffer solution of probe L, we observed that the fluorescence intensity of probe L at 640 nm ranged from 12 to 36×10 -6 mol / L H2S concentration range has a good linear relationship ( Figure 7 ). Therefore, we calculated that the detection limit of probe L for H2S is 3.09×10 -6 mol / L (according to the equation LOD = 3S / k, where S is the standard deviation of the blank solution, k is the slope of the standard curve, R 2 >0.99), the detection limit of probe L was low, indicating that probe L can sensitively identify H2S.

[0074] Response time test of fluorescent probe L to H2S:

[0075] In the DMF:PBS=3:7 (v / v, pH=7.4) buffer solution of probe L, 6 times HS - Then the fluorescence intensity changes at different times were tested. Figure 8 It can be seen that the fluorescence intensity of the probe gradually increases with time, reaching the highest value at around 170 minutes and showing a stable trend, indicating that the recognition of H2S by probe L can be completed within 170 minutes and has the ability to respond quickly.

[0076] Test on the effect of pH on the response of fluorescent probe L to H2S:

[0077] In the DMF:PBS=3:7 (v / v, pH=7.4) buffer solution of probe L, we tested the effect of different pH on the recognition of H2S by probe L and verified the practicality of probe L in biological and environmental systems, such as Fig. 9 The fluorescence intensity of probe L in DMF / PBS (3 / 7, v / v, pH=7.4) buffer solution remained basically unchanged within the pH range of 2 to 10. - After the reaction, the fluorescence was significantly enhanced in the pH range of 7 to 11, indicating that the probe L has a wide pH range of application, including the biologically relevant range. - Therefore, probe L can be potentially used to detect H2S in biological systems.

[0078] Fluorescent probe L detects H2S in actual water samples:

[0079] In order to test the practical applicability of probe L to H2S, we first explored the potential application of L in real water samples. Each water sample was spiked with different concentrations of HS- ions, and then probe L (1×10 -6 mol / L) in these natural water samples, the fluorescence intensity and the addition of HS - Concentration (0~6×10 -5 mol / L) show a good linear relationship, such as Fig.10 Therefore, probe L has potential application in the quantitative detection of H2S in environmental systems.

[0080] Fluorescent probe L imaging in living cells:

[0081] To further examine the application of L in living cells, MCF-7 cells were incubated with L (1×10 -6 mol / L) at 37°C for 30 minutes, and then washed three times with PBS buffer. In the dark field, only a faint red fluorescence ( Fig.11 When MCF-7 cells pretreated with L were exposed to different concentrations of H2S (10, 50 and 100×10 -6 mol / L) for further 30 minutes, the brightness of red fluorescence under dark field will increase with the increase of HS - The concentration gradually increases ( Fig.11 These results suggest that probe L can easily cross the membrane barrier, permeate into MCF-7 cells, and rapidly sense intracellular HS - Bright-field images of the treated cells before and after treatment did not show any morphological changes, indicating that the MCF-7 cells were still viable, opening up the way for potential biomedical applications of probe L.

[0082] It should be noted that the present invention is not limited to the above-mentioned embodiments. The above-mentioned embodiments are only preferred examples, and the embodiments having the same structure as the technical idea and exerting the same effect within the technical solution of the present invention are all included in the technical scope of the present invention. In addition, without departing from the scope of the main purpose of the present invention, various modifications that can be thought of by those skilled in the art to the embodiments, and other methods of combining some of the constituent elements in the embodiments and constructing them should also be considered within the scope of the present invention.

Claims

1. A long-wavelength H2S fluorescent probe, characterized in that: The structural formula is as follows:

2. A method for preparing a long-wavelength H2S fluorescent probe, characterized in that: The following steps are involved: (1) dissolving 3-hydroxy-3-methyl-2-butanone and malononitrile in ethanol, then adding dry ethanol to heat and reflux, cooling after the reaction is completed, filtering the precipitated solid, washing the solid with cold ethanol, and then dissolving the washed solid in ethanol for recrystallization, cooling after completion, filtering the precipitated solid, and obtaining pure compound 2; (2) dissolving the obtained compound 2 and 4-diethylamino salicylaldehyde in ethanol, heating again under alkaline conditions for reflux reaction, and after the reaction is completed, drying the obtained crude product and purifying it by thin layer column chromatography to obtain compound 1; (3) Compound 1, 2,4-dinitrofluorobenzene and anhydrous potassium carbonate are dissolved in a polar aprotic solvent and reacted by stirring at room temperature. After the reaction is completed, the solid is filtered out and then spin-dried to obtain a crude product. The crude product is purified by thin layer column chromatography to obtain a H2S fluorescent probe.

3. The preparation method according to claim 2, characterized in that : Sodium ethoxide is also added as a catalyst in the reflux reaction in step (1), and the mass ratio of 3-hydroxy-3-methyl-2-butanone to malononitrile to sodium ethoxide is (8-12):(10-15):

1.

4. The preparation method according to claim 2, characterized in that: In both the thin layer column chromatography purification in step (2) and step (3), ethyl acetate and petroleum ether are used as eluents for separation; the volume ratio of ethyl acetate to petroleum ether in step (2) is 1:5, and the volume ratio of ethyl acetate to petroleum ether in step (3) is 1:

3.

5. The preparation method according to claim 4, characterized in that: In the step (2), the mass ratio of compound 2 to 4-diethylaminosalicylaldehyde is 5:(3-4).

6. The preparation method according to claim 2, characterized in that: In the step (3), the mass ratio of compound 1 to 2,4-dinitrofluorobenzene and anhydrous potassium carbonate is (1-2): (1-1.5):

1.

7. The preparation method according to claim 2, characterized in that: In the step (3), the polar aprotic solvent is N,N-dimethylformamide.

8. An application of a long-wavelength H2S fluorescent probe, characterized in that: The H2S fluorescent probe according to any one of claims 1 to 8 is used to detect H2S in a buffer solution of DMF and PBS at a pH of 7 to 11 and a volume ratio of 3:

7.

9. An application of a long-wavelength H2S fluorescent probe, characterized in that: The H2S fluorescent probe described in any one of claims 1 to 8 is used to detect H2S in an actual water sample.

10. An application of a long-wavelength H2S fluorescent probe, characterized in that: The H2S fluorescent probe according to any one of claims 1 to 8 is used to detect HS in MCF-7 living cells - Perform detection imaging.

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