A ratiometric fluorescent probe for detecting SO2 derivatives and its synthesis method and application

Abstract

The present invention discloses a ratiometric fluorescent probe for detecting SO2 derivatives and its synthesis method and application. The fluorescent probe has the molecular formula C 41 H 44 ClN5O8. The fluorescent probe provided by the present invention can specifically recognize SO2 derivatives and undergo nucleophilic addition reaction, thereby blocking the ICT effect inside the oxonium structure, and then blocking the FRET effect, showing a transition from red fluorescence to green fluorescence. The optical properties of the probe are excellent: it has a 191nm emission band gap, which can effectively avoid the occurrence of fluorescence overlap; high specificity to avoid interference from other substances; and excellent sensitivity, with a detection limit of 64μM. In addition, when the Na2SO3 concentration is in the range of 0-50μM, it is proportional to the fluorescence ratio of the probe, R 2 >0.99, which means that the Na2SO3 concentration can be quantitatively calculated based on the fluorescence ratio within this range.

Description

Technical Field

[0001] The present invention belongs to the technical field of organic small molecule ratiometric fluorescent probes, and particularly relates to a ratiometric fluorescent probe for detecting SO2 derivatives, a synthesis method thereof, and applications thereof. Background Art

[0002] This section merely provides background information related to the present disclosure and is not necessarily prior art.

[0003] Due to the rapid development of modern industry, SO2 concentrations in the environment have increased dramatically, causing increasingly severe pollution. SO2 is also produced within organisms, originating from the enzymatic activity of mitochondria, which catalyzes the decomposition of sulfur-containing amino acids. Research has found that SO2 is a gaseous signaling molecule that plays a key role in regulating the cardiovascular system and maintaining redox balance. Therefore, SO2 concentration can be used as an indicator for screening for cardiovascular disease.

[0004] Methods for detecting SO2 concentration include colorimetry, titration, and electrochemistry. However, these methods cannot achieve non-destructive detection of SO2 concentration in living organisms. Fluorescent probes effectively address this shortcoming, ensuring real-time monitoring of SO2 concentration while avoiding cell damage.

[0005] To date, a variety of probes for SO₂ detection have been reported, including turn-on fluorescent probes based on changes in single-channel fluorescence intensity and ratiometric probes based on dual-channel fluorescence. Ratiometric probes are generally considered superior to single-channel fluorescent probes because their fluorescence intensity is often affected by factors such as substrate concentration, instrument fluctuations, and background fluorescence, whereas ratiometric probes can minimize the effects of these destabilizing factors. The development of new probes with low toxicity, good water solubility, and high stability remains a priority. Summary of the Invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to address the deficiencies of the prior art and provide a ratiometric fluorescent probe for detecting SO2 derivatives and its synthesis method and application.

[0007] In order to solve the above technical problems, the present invention discloses the following technical solutions:

[0008] In the first aspect, the present invention discloses a ratiometric oxonium fluorescent probe as shown in Formula I, which is labeled as FJ-A1 and has a molecular formula of C 41 H 44 ClN5O8;

[0009]

[0010] In a second aspect, the present invention discloses a method for preparing the ratiometric oxonium fluorescent probe described in the first aspect, comprising the following steps:

[0011] (1) reacting o-hydroxybenzaldehyde with acetophenone, and after the reaction is completed, adding perchloric acid and stirring to obtain an oxonium intermediate (1,4-diethyl-7-(4-(piperazin-1-yl)phenyl)-1,2,3,4-tetrahydropyrano[2,3-g]quinoxalin-6-ium perchlorate) as shown in Formula II;

[0012] (2) reacting the oxonium intermediate represented by formula II with coumarin 343 to obtain the ratiometric oxonium fluorescent probe represented by formula I;

[0013]

[0014] In some embodiments, step (1) is specifically to stir and dissolve o-hydroxybenzaldehyde, acetophenone and concentrated sulfuric acid, pour the resulting reaction solution into water, add perchloric acid, precipitate a black solid, continue stirring, filter, and dry to obtain the oxonium intermediate shown in formula II.

[0015] In step (1), the molar ratio of o-hydroxybenzaldehyde to acetophenone is 1:0.8-1.2, preferably 1:1.

[0016] In step (1), the mass volume ratio of o-hydroxybenzaldehyde to concentrated sulfuric acid is 1.8 g:15-25 mL.

[0017] In step (1), the reaction temperature is 85-95°C, preferably 90°C.

[0018] In step (1), the reaction time is 5-7 hours, such as 6 hours.

[0019] In step (1), the mass volume ratio of o-hydroxybenzaldehyde to water is 1.8g:180-220mL.

[0020] In step (1), the mass volume ratio of o-hydroxybenzaldehyde to perchloric acid is 1.8 g:4-6 mL.

[0021] In step (1), the stirring time is 1-3 hours, such as 2 hours.

[0022] In some embodiments, step (2) is specifically to replace the inert gas with coumarin 343, a condensing agent, a base, and a solvent, and then add the oxonium intermediate shown in Formula II to react after stirring. After the reaction is completed, the reaction is quenched and extracted with DCM, and the organic phases are combined. The obtained organic phases are purified by column chromatography to obtain the ratiometric oxonium fluorescent probe shown in Formula I.

[0023] In step (2), the condensing agent includes 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; the molar ratio of coumarin 343 to the condensing agent is 1.4:1.5-1.86, preferably 1.4:1.6-1.76.

[0024] In step (2), the base includes 4-dimethylaminopyridine; the molar ratio of coumarin 343 to the base is 1.4:0.15-0.41, preferably 1.4:0.2-0.36.

[0025] In step (2), the reaction solvent includes N,N-dimethylformamide; the molar volume ratio of coumarin 343 to the solvent is 1.4 mmol:8-12 mL.

[0026] In step (2), the molar ratio of the coumarin 343 to the oxonium intermediate represented by formula II is 1:0.8-1.4, such as 1:1.2.

[0027] In step (2), the stirring is performed at room temperature for 0.3-0.7 h.

[0028] In step (2), the reaction time is 6-10 hours, such as 8 hours.

[0029] In step (2), after the reaction is completed, water is added to quench the reaction; DCM is extracted 4-6 times, each time with 40-60 mL; the organic phases are combined, concentrated, and purified by column chromatography (DCM / MeOH=75-85 / 1, v / v) to obtain the ratiometric oxonium fluorescent probe shown in Formula I.

[0030] In a third aspect, the present invention discloses the use of the ratiometric oxonium fluorescent probe described in the first aspect in detecting SO2 derivatives, as well as the use of the probe in preparing detection reagents or test papers for detecting SO2 derivatives.

[0031] The SO2 derivatives described in the present invention include sulfites and bisulfites.

[0032] The ratiometric oxonium fluorescent probe provided by the present invention can be used as a medium for the neutral polar solvent PBS / ethanol to achieve visual and highly sensitive detection of SO2 derivatives. The ratiometric oxonium fluorescent probe can specifically identify SO2 derivatives and undergo a nucleophilic addition reaction on the unsaturated double bond of the oxonium, causing the characteristic hydrogens Ha and Hb of the probe to move upfield from 8.06 ppm and 6.88 ppm to 4.36 ppm and 5.51 ppm, corresponding to Hc and Hd ( Figure 7), thereby blocking the ICT effect inside the oxonium structure and then blocking the FRET effect, showing a transition from red fluorescence to green fluorescence. The optical properties of this probe are excellent: it has a 191nm emission band gap, which can effectively avoid the occurrence of fluorescence overlap; high specificity to avoid interference from other substances; and excellent sensitivity, with a detection limit of 64μM. In addition, when the Na2SO3 concentration is in the range of 0-50μM, the fluorescence ratio (I 494 / I 685 ) are proportional to each other, R 2 >0.99, which means that the concentration of Na2SO3 can be quantitatively calculated based on the fluorescence ratio within this range. The fluorescent probe can be successfully used in the visualization experiment of HepG2 cells as a potential tool for detecting SO2 derivatives.

[0033] Beneficial effects:

[0034] 1. This invention designs a ratiometric oxonium fluorescent probe, FJ-A1, which is an ideal sensor for the rapid and sensitive detection of SO2 derivatives. This fluorescent probe reacts with SO2 derivatives under very mild conditions using a medium-polarity solvent, resulting in a strong sensing process with rapid response, high sensitivity, and good specificity, enabling real-time quantitative detection of SO2 derivatives.

[0035] 2. When the ratiometric oxonium fluorescent probe FJ-A1 synthesized in the present invention can specifically detect SO2 derivatives - (bisulfite), nucleophilic addition occurs, blocking the ICT effect inside the receptor, resulting in the destruction of FRET and ultimately showing donor green fluorescence. It has broad application prospects in the development of visual detection and test strips.

[0036] 3. Compared with existing fluorescent probe technologies, the synthesis method of the ratiometric oxonium fluorescent probe FJ-A1 in the present invention is simple, the reaction temperature is low, the synthesis process is fast, the operation steps are simple, and the yield is considerable. It is environmentally friendly. The synthesized ratiometric oxonium fluorescent probe FJ-A1 can realize micromolar level monitoring of SO2 derivatives and has good development prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more apparent.

[0038] Figure 1 This is a synthetic route for the ratiometric oxonium fluorescent probe FJ-A1 in Example 1 of the present invention.

[0039] Figure 2 Graph showing the fluorescence emission spectrum of the donor and the ultraviolet absorption spectrum of the acceptor in Example 2 of the present invention.

[0040] Figure 3 This is the UV-visible titration spectrum of probe FJ-A1 (10 μM) before and after the reaction with Na2SO3 (0-250 μmol) in the performance test of the present invention.

[0041] Figure 4 This is the fluorescence titration spectrum of the reaction between the ratiometric oxonium fluorescent probe FJ-A1 and Na2SO3 (0-3mmol) in the performance test of the present invention, where Figure 4 The wavelength range of a is 420-900nm, Figure 4 The b wavelength range is 630-800nm.

[0042] Figure 5 This is the linear relationship between the ratiometric oxonium fluorescent probe FJ-A1 and the concentration of Na2SO3 in the performance test of the present invention.

[0043] Figure 6 This is the selectivity experiment of the ratiometric oxonium fluorescent probe FJ-A1 in the performance test of the present invention, (a) fluorescence intensity, (b) fluorescence ratio.

[0044] Figure 7 The reaction mechanism of ratiometric oxonium fluorescent probe FJ-A1 with Na₂SO₃ in the performance test of this invention. (a) The characteristic H₁ and H₂ NMR shifts of the probe; (b) The characteristic H₁ and H₂ NMR shifts of probe 3 after reaction with Na₂SO₃.

[0045] Figure 8 This is a fluorescence confocal image of the ratiometric oxonium fluorescent probe FJ-A1 and probe 3 (5 μM) recognizing Na 2 SO 3 in HepG-2 cells in the performance test of the present invention. DETAILED DESCRIPTION

[0046] The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the contents described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0047] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

[0048] The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0049] The drying solvents used in the following examples are all molecular sieves 4A (sodium-type A molecular sieve) or molecular sieves 3A (potassium-type A molecular sieve).

[0050] 1H NMR spectra were recorded on a Bruker Avance 400 NMR spectrometer using DMSO-d6 as solvent.

[0051] Upfield chemical shifts are reported in parts per million based on internal TMS (trimethylsilane) reference data.

[0052] The coupling constant (J) is expressed in Hertz (Hz), and the spin states are denoted by s (singlet), d (doublet), t (triplet), and m (multiplet).

[0053] Column chromatography used thick-walled glass columns and silica gel (200-300 mesh).

[0054] Thin layer chromatography (TLC) was performed using commercially available 0.25 mm silica gel plates and visualized using UV light.

[0055] The UV absorption spectra of the solutions were obtained using a UNICO UV-4802 UV-visible-near-infrared spectrophotometer.

[0056] The fluorescence spectra were measured using LSM880 fluorescence spectrophotometer.

[0057] Mass spectra were recorded on a MicroTOF Bruker high performance liquid chromatography-mass spectrometer.

[0058] Example 1

[0059] This embodiment provides a ratiometric oxonium fluorescent probe, the synthesis method of which is as follows: Figure 1 As shown, the following steps are included:

[0060] S1. Add o-hydroxybenzaldehyde (1.8 g, 7.7 mmol), acetophenone (1.57 g, 7.7 mmol), and 98% concentrated sulfuric acid (20 mL) to a 25 mL single-necked flask, stir to dissolve, and heat to 90°C for 6 h. After the reaction, pour the reaction solution into 200 mL of water and add 5 mL of perchloric acid dropwise to precipitate a black solid. Continue stirring at 20-30°C for 2 h, filter, and dry to obtain 1.28 g of the oxonium intermediate 1,4-diethyl-7-(4-(piperazin-1-yl)phenyl)-1,2,3,4-tetrahydropyrano[2,3-g]quinoxalin-6-ium perchlorate, in a yield of 33%. 1H NMR (400MHz, DMSO-d6) δ8.29(d,J=8.3Hz,1H),8.10(d,J=9.1Hz,2H),7.84(d,J=8.3Hz,1H),7.22(s,1H),7.15 (d,J=8.8Hz,2H),6.86(s,1H),3.66(dt,J=10.8,5.0Hz,8H),3.54–3.31(m,8H),1.20(dt,J=38.8,7.0Hz,7H). 13 C NMR(101MHz,DMSO-d6)δ161.12,154.84,152.62,147.15,141.76,136.41,128.58,119.89,119.34, 114.57,108.13,102.57,94.41,45.14,43.69,43.20,42.54,10.82,9.41.HR-MS(ESI+):[C25H31N4O + ]Predicted value 403.2492, test value 403.2492.

[0061] S2. Coumarin 343 (400 mg, 1.40 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (323 mg, 1.68 mmol), 4-dimethylaminopyridine (34 mg, 0.28 mmol), and N,N-dimethylformamide (10 mL) were added sequentially to a 25 mL three-necked flask. The atmosphere was purged with nitrogen three times and stirred at room temperature for 0.5 h. The oxonium intermediate (775 mg, 1.54 mmol) was then added and the reaction continued for 8 h. After completion, the reaction was quenched with 10 mL of water and extracted with DCM five times, each time with 50 mL. The organic phases were combined, concentrated, and purified by column chromatography (DCM / MeOH = 80 / 1, v / v) to obtain 460 mg of probe FJ-A1, with a yield of 42.6%. 1H NMR (400MHz, DMSO-d6), δ: 8.30 (d, J = 8.4Hz, 1H); 8.11-8.07 (m, 1H); 8.06 (s, 1H); 7.90 (s, 1H); 7.82 (d, J=8.4Hz, 1H); 7.26-7.18 (m, 1H); 7.12-7.03 (m, 3H); 6.88 (s, 1H); 3.70 (q, J= 7.6, 6.5Hz, 6H); 3.59-3.42 (m, 8H); 3.41-3.36 (m, 2H); 3.32-3.24 (m, 4H); 2.70 (dt, J = 14. 7, 6.2Hz, 4H); 1.86 (tq, J = 11.9, 6.1Hz, 4H); 1.25 (t, J = 7.1Hz, 3H); 1.16 (t, J = 7.0Hz, 3H). 13 C NMR (101MHz, DMSO-d6), δ: 165.01, 162.08, 159.06, 155.08, 153.65, 152.00, 147. 35,147.17,144.95,136.75,129.19,126.40,119.91,119.13,118.67,114.82,114 .48,108.51,107.35,105.54,103.20,99.99,94.90,49.84,49.34,47.42,47.24,4 5.60,43.73,27.28,21.20,20.28,20.14,11.27,9.89.HR-MS(ESI+):[C41H44N5O4 + ]Predicted value 670.3388, test value 670.33815.

[0062] Example 2

[0063] This example conducts an optical experiment on the ratiometric oxonium fluorescent probe in Example 1, including the following steps:

[0064] S1. Take an appropriate amount of the probe and dissolve it in anhydrous ethanol, then dilute it to 20 μM with PBS buffer (10 mM, pH=7.4) as a stock solution.

[0065] S2. Dissolve an appropriate amount of Na2SO3 in PBS buffer (containing 20% ​​ethanol) to prepare a 200 μM solution, and then dilute it to a series of different concentrations.

[0066] S3. Dissolve various analytes, such as sodium chloride, hydrazine hydrate, and sodium sulfide, in PBS buffer to a concentration of 1 mM for optical testing. The excitation and emission slits of the fluorescence spectrophotometer are both 10 nm.

[0067] Probe design

[0068] The oxonium structure can undergo a specific addition reaction with (bisulfite), resulting in the quenching of near-infrared fluorescence and the display of green fluorescence. In order to study the fluorescence quantum yield of the probe FJ-A1, the donor structure and the acceptor structure were dissolved in anhydrous ethanol to a concentration of 5mM, and the fluorescence spectrum of the donor and the ultraviolet absorption spectrum of the acceptor were collected. Figure 2 As shown, the two spectra have a large overlap, which is one of the necessary conditions for FRET probes. Subsequently, the acceptor and donor were mixed and prepared into ethanol solutions with a concentration of 5mM each. The fluorescence spectra were collected and the fluorescence quantum yield formula was used. The FRET efficiency of FJ-A1 is 26.6%. The emission wavelength of FJ-A1 blue-shifts from 685nm to 494nm before and after the reaction, with an emission band as wide as 191nm, ensuring a clear fluorescence change and effectively avoiding the occurrence of fluorescence overlap.

[0069] Optical response

[0070] The excitation wavelength of the probe was determined by UV-visible titration experiment, and the concentration of FJ-A1 was 10 μM. Figure 3 As shown in the figure, in a gradient Na₂SO₃ solution, the UV absorption of FJ-A1 near 410 nm decreases with increasing Na₂SO₃ concentration, ultimately disappearing. The absorption intensity near 645 nm also decreases, and the intensity at 560 nm shows no significant change. Considering that shorter excitation wavelengths are more reasonable, 410 nm was selected as the excitation wavelength for the probe.

[0071] Under 410nm excitation, the maximum emission wavelength of FJ-A1 is located at 685nm. Figure 4 As shown in the figure, after treatment with different concentrations of Na2SO3, the fluorescence spectrum showed expected changes: the 685nm fluorescence decreased while the 494nm fluorescence increased sharply. According to the curve of fluorescence change ratio and Na2SO3 concentration, as shown in the figure, Figure 5 As shown in the results, it is obvious that the concentration of Na2SO3 in the range of 0-50μM is related to I 494 / I 685 The fluorescence ratio is proportional to each other, R 2 >0.99, which means that within this range, the concentration of Na2SO3 can be quantitatively determined based on the fluorescence intensity, with a detection limit of 64 μM.

[0072] Selective research

[0073] Anions, cations, active sulfur and other substances were introduced into the specificity experiment, especially active sulfur, which is a potential interfering substance. Each analyte was prepared into a solution with a concentration of 1 mM, and thoroughly mixed with an equal volume of the probe stock solution in S1. Fluorescence spectroscopy was tested after 5 minutes. Figure 6 The results showed that only HS - It can cause a slight fluorescence enhancement of FJ-A1, but it is similar to SO3 2- / HSO3 - In comparison, the fluorescence change was not obvious; as for other substances, none of them triggered the fluorescence response of the probe, which verified the good specificity of FJ-A1.

[0074] Example 3

[0075] This example uses the ratiometric oxonium fluorescent probe in Example 1 to apply to a HepG-2 cell imaging experiment, including the following steps:

[0076] S1. HepG-2 cells were cultured at 37°C in an atmosphere containing 5% CO2 using MEM medium. Cells were incubated in medium containing 5 μmol / L of the probe for 1 hour, then transferred to medium containing various concentrations of Na2SO3 (25, 50, and 200 μmol / L) and incubated for another hour.

[0077] S2. Rinse with PBS buffer and image the cells on a confocal laser microscope. Under excitation at 410 nm, collect green fluorescence from 450 nm to 600 nm and red fluorescence from 640 nm to 780 nm.

[0078] As attached Figure 8 As shown, the cells exhibit strong fluorescence in both the red and green channels. As the Na₂SO₃ concentration increases, the intensity of the red channel gradually decreases, while the green fluorescence increases significantly. When the Na₂SO₃ concentration reaches 200 μM, the red fluorescence almost disappears, while the green fluorescence intensity reaches its maximum. This demonstrates that FJ-A1 can be successfully used in cell imaging experiments, demonstrating distinct fluorescence changes.

[0079] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A ratiometric oxonium fluorescent probe as shown in Formula I; I。 2. The method for preparing the ratiometric oxonium fluorescent probe according to claim 1, wherein: The following steps are involved: (1) reacting o-hydroxybenzaldehyde with acetophenone, and after the reaction is complete, adding perchloric acid and stirring to obtain an oxonium intermediate shown in formula II; (2) reacting the oxonium intermediate represented by formula II with coumarin 343 to obtain the ratiometric oxonium fluorescent probe represented by formula I; II.

3. The preparation method according to claim 2, characterized in that In step (1), the molar ratio of o-hydroxybenzaldehyde to acetophenone is 1:0.8-1.

2.

4. The preparation method according to claim 2, characterized in that In step (1), the reaction temperature is 85-95° C. and the reaction time is 5-7 h.

5. The preparation method according to claim 2, characterized in that In step (1), the mass volume ratio of o-hydroxybenzaldehyde to perchloric acid is 1.8 g:4-6 mL; and the stirring time is 1-3 h.

6. The preparation method according to claim 2, characterized in that In step (2), the molar ratio of the coumarin 343 to the oxonium intermediate represented by formula II is 1:0.8-1.

4.

7. The preparation method according to claim 2, characterized in that In step (2), the reaction further comprises a condensing agent and a base.

8. The preparation method according to claim 7, characterized in that The condensing agent includes 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

9. The preparation method according to claim 7, characterized in that The base includes 4-dimethylaminopyridine.

10. The preparation method according to claim 7, characterized in that In step (2), coumarin 343, a condensing agent, a base and a solvent are stirred and then an oxonium intermediate represented by formula II is added for reaction.

11. The preparation method according to claim 2, characterized in that In step (2), the solvent for the reaction includes N,N-dimethylformamide.

12. Use of the ratiometric oxonium fluorescent probe according to claim 1 in the preparation of a detection reagent or test paper for detecting SO2 derivatives.

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