Fluorescent probe for detecting sulfur dioxide derivatives and viscosity in an organic solvent-free system and application thereof

Abstract

The application discloses a double-response ratio fluorescent probe for detecting sulfur dioxide derivatives and viscosity in an organic solvent-free system, which is composed of a coumarin derivative fluorophore (energy donor), piperazine (linking group) and (E)-5-formyl-1,3,3-trimethyl-2-(2-(2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinolin-9-yl)vinyl)-3H-indol-1 iodine salt (energy acceptor) three parts, and a chemical structural formula is shown as formula (I). The probe can be used for high selectivity with sulfur dioxide derivatives in the test system without organic solvents, and with the increase of the concentration of sulfur dioxide derivatives, the fluorescence emission intensity gradually increases at 487 nm and gradually weakens at 625 nm; the ratio (I 487 / I 625 ) of the two is linearly related to the concentration of sulfur dioxide derivatives in a certain range. Meanwhile, the probe can also detect the change of viscosity. It is indicated that the probe has wide application in the detection of samples containing sulfur dioxide derivatives or samples with different viscosities, and is expected to play a role in clinical medical diagnosis and treatment.

Description

Technical Field

[0001] This invention relates to a fluorescent probe for testing systems that do not require organic solvents and its applications, particularly to a dual-response ratiometric fluorescent probe based on fluorescence resonance energy transfer mechanism, using a coumarin derivative as the donor and (E)-5-formyl-1,3,3-trimethyl-2-(2-(2,3,6,7-tetrahydro-1H,5H-pyridin[3,2,1-ij]quinolin-9-yl)vinyl)-3H-indole-1 iodide as the acceptor, for detecting sulfur dioxide derivatives and viscosity in solvent-free systems, and its applications; belonging to the field of organic small molecule fluorescent probe technology. Background Technology

[0002] On the one hand, sulfur dioxide (SO2), whether in gaseous form or in the form of HSO3 - SO3 2- Sulfur dioxide, in its various forms, pollutes the environment. On the other hand, sulfur dioxide derivatives are used as additives in food and wine. While sulfur dioxide derivatives have physiological functions, abnormal concentrations can trigger various diseases. Real-time monitoring of endogenous HSO3 in organisms is crucial. - SO3 2- The content helps in the study of HSO3 - SO3 2- Intracellular viscosity plays a vital role in physiological and pathological processes. It influences biological signal transduction and is closely related to physiological and pathological conditions; for example, abnormal viscosity is associated with Alzheimer's disease. Therefore, detecting intracellular viscosity is essential for studying its biological functions.

[0003] Fluorescent probe research has seen rapid advancements in recent years, with fluorescent probes possessing high selectivity, high sensitivity, and low detection limits being particularly promising. As one of the research hotspots, their application in detecting HSO3- is... - SO3 2-Fluorescent probes with high viscosity have attracted much attention [Chem. Soc. Rev., 2018, 47, 8842-8880; Chem. Eur. J. 2022, e202202366]. Fluorescent probes are mainly of two types: single-emission and dual-emission ratiometric. Ratiometric fluorescent probes can achieve self-calibration through the ratio of the intensities of the two emission peaks, exhibiting good stability and strong anti-interference ability, showing significant advantages over single-emission fluorescent probes. Ratiometric probes designed based on intramolecular charge transfer mechanisms often suffer from shortcomings such as small peak spacing, mutual interference, and poor accuracy; ratiometric fluorescent probes based on fluorescence resonance energy transfer (FRET) mechanisms have superior performance. A probe consists of three parts: an energy donor, an energy acceptor, and a linker. By exciting the donor, energy is transferred to the acceptor, and the probe exhibits enhanced acceptor fluorescence. Based on the recognition site and mechanism, FRET systems can be mainly divided into two types of templates. One type of FRET template involves the probe itself being an FRET system. After recognizing the target species, the FRET process is blocked, and the receptor cannot accept the donor's energy. The fluorescence emission intensity of both the donor and receptor gradually changes with the concentration of the target species. The other type of template involves the probe itself being an FRET-blocking system. After reacting with the target species, the FRET process gradually begins, and the receptor accepts the donor's energy. The fluorescence emission intensity of both the donor and receptor gradually changes with the concentration of the target species. Therefore, the ratio of the fluorescence intensity of the donor and receptor exhibits a functional relationship with the concentration of the target species within a certain range. Currently, many FRET-based fluorescent probes suffer from drawbacks such as low energy transfer efficiency, small distance between the two emission wavelengths, and long response times. In particular, the reported fluorescent probes have poor water solubility, often requiring organic solvents in the testing system for spectral analysis. Therefore, there is an urgent need to develop new fluorophores to construct novel FRET-based fluorescent probes to better meet application requirements. A search revealed few reports on dual-response ratiometric fluorescent probes based on fluorescence resonance energy transfer mechanisms for the detection of sulfur dioxide derivatives and viscosity in solvent-free systems, and their applications. In particular, there is an urgent need to develop detection systems without organic solvents. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a ratiometric fluorescent probe with dual response to detect sulfur dioxide derivatives and viscosity under organic solvent-free detection system conditions, and its application.

[0005] The present invention describes a dual-response ratiometric fluorescent probe for detecting sulfur dioxide derivatives and viscosity in an organic solvent-free system, characterized in that: the ratiometric fluorescent probe comprises a coumarin derivative fluorophore as an energy donor, piperazine as a linking group, and (E)-5-formyl-1,3,3-trimethyl-2-(2-(2,3,6,7-tetrahydro-1H,5H-pyridin[3,2,1-ij]quinoline-9-yl)vinyl)-3H-indyl The indole-1-iodide salt is composed of three parts as an energy acceptor; its chemical name is (E)-5-(4-(7-(diethylamino)-2-oxo-2H-chroman-3-carbonyl)piperazin-1-carbonyl)-1,3,3-trimethyl-2-(2-(2,3,6,7-tetrahydro-1H,5H-pyridin[3,2,1-ij]quinolin-9-yl)vinyl)-3H-indole-3-iodide, and its chemical structural formula is shown in formula (I):

[0006]

[0007] The preparation method of the above-mentioned dual-response ratiometric fluorescent probe for detecting sulfur dioxide derivatives and viscosity in organic solvent-free systems is as follows: 5-carboxy-1,2,3,3-tetramethyl-3H-indole-1-iodide salt is condensed with 2,3,6,7-tetrahydro-1H,5H-pyridin[3,2,1-ij]quinoline-9-carboxaldehyde to obtain (E)-5-carboxy-1,3,3-trimethyl-2-(2-(2,3,6,7-tetrahydro-1H,5H-pyridin[3,2,1-ij]quinoline-9-yl)vinyl)-3H-indole-1-iodide salt, which is then reacted with 7-(diethylamino)-3-(piperazin-1-carbonyl)-2H-chroman-2-one to obtain the dual-response ratiometric fluorescent probe (abbreviated as CIJ) for detecting sulfur dioxide derivatives and viscosity in organic solvent-free systems.

[0008] The chemical reaction formula for the preparation of the ratiometric fluorescent probe is as follows:

[0009]

[0010] The present invention relates to the application of a dual-response ratiometric fluorescent probe for detecting sulfur dioxide derivatives and viscosity in organic solvent-free systems in the detection of samples containing sulfur dioxide derivatives.

[0011] The present invention relates to the application of a dual-response ratiometric fluorescent probe for detecting sulfur dioxide derivatives and viscosity in organic solvent-free systems in the detection of samples with different viscosities.

[0012] Wherein: the samples containing sulfur dioxide derivatives or of different viscosities refer to cultured cells or solutions of different viscosities.

[0013] The present invention provides a dual-response ratiometric fluorescent probe for detecting sulfur dioxide derivatives and viscosity in solvent-free systems. In the absence of sulfur dioxide derivatives, the energy donor is excited and transfers energy to the energy acceptor, resulting in enhanced acceptor fluorescence. Upon reaction with sulfur dioxide derivatives, new addition products are generated, altering the structure of the energy acceptor, such as... Figure 1 As shown, the FRET energy transfer process is blocked, and the excited energy donor exhibits donor fluorescence emission. The fluorescence intensity at both emission wavelengths changes with the concentration of the sulfur dioxide derivative; thus, ratiometric detection of the sulfur dioxide derivative is achieved. Because the CIJ receptor structure contains rotatable chemical bonds, the TICT process is inhibited, resulting in weak fluorescence intensity. When the viscosity of the recognition system increases, the TICT process is suppressed, and the fluorescence intensity increases, as shown... Figure 1 As shown.

[0014] Specifically: Prepare the test solution (HEPES buffer, pH = 7.4) of the dual-response ratiometric fluorescent probe for detecting sulfur dioxide derivatives and viscosity in an organic solvent-free system as described in this invention, by adding a certain amount of aqueous solutions of reactive oxygen species, reactive nitrogen species, and metal ions, such as: F - ,Cl - ,Br - ,I - Cys, Hcy, GSH, HS - H2O2, HClO, CH3CO 2- SO4 2- S2O3 2- NO3 - NO2 - CO3 2- SCN - Mg 2+ Ca 2+ Fluorescence testing of the above solutions showed that the probe of the present invention has good selectivity and anti-interference ability for sulfur dioxide derivatives. (See attached image.) Figure 2 .

[0015] Under 417 nm excitation light, the fluorescence intensity of the fluorescent probe described in this invention gradually increases at 487 nm and gradually decreases at 625 nm with increasing concentration of sulfur dioxide derivative; the ratio of these two intensities shows a linear relationship with the concentration of sulfur dioxide derivative within a certain range. This confirms that the probe can quantitatively detect low concentrations of sulfur dioxide derivative. Figure 3 .

[0016] In HepG2 live cells with the above-mentioned probe CIJ, the changes in cell fluorescence microscopy were observed using laser scanning confocal microscopy in cells without GSH / Na2S2O3 and with GSH / Na2S2O3 (GSH / Na2S2O3: promotes the endogenous production of sulfur dioxide derivatives in cell metabolism). It can be seen that cells without GSH / Na2S2O3 showed stronger red channel fluorescence and weaker blue channel fluorescence; in the experimental group (with GSH / Na2S2O3), the red channel fluorescence was weaker than the control group, while the blue channel fluorescence was significantly stronger; the ratio of the blue channel fluorescence intensity to the red channel fluorescence intensity changed very significantly. Figure 4 .

[0017] In L-O2 live cells with the above-mentioned probe CIJ, the changes in cell fluorescence microscopy were observed using laser scanning confocal microscopy for cells with added GSH / Na2S2O3 and NaHSO3. It can be seen that the experimental group with added GSH / Na2S2O3 showed stronger red channel fluorescence and weaker blue channel fluorescence, with no significant difference between the two and the control group; the cells with added Na2HSO3 showed weaker red channel fluorescence and stronger blue channel fluorescence; the ratio of the statistical values ​​of blue channel fluorescence intensity to red channel fluorescence intensity changed significantly. Figure 5 .

[0018] HeLa cells were incubated with nystatin (10 μM) for half an hour, followed by incubation with CIJ (5 μM) for 1 hour. Nystatin increased cell viscosity, and the changes in the statistical value of fluorescence intensity in the red channel were very significant when observed using a laser scanning confocal microscope. Figure 6 .

[0019] In summary, the ratiometric fluorescent probe disclosed in this invention for detecting sulfur dioxide derivatives and viscosity in an organic solvent-free system exhibits good water solubility, eliminating the need for organic solvents in the test system. On one hand, it reacts with sulfur dioxide derivatives with high selectivity; as the concentration of the sulfur dioxide derivative increases, its fluorescence emission intensity gradually increases at 487 nm and gradually decreases at 625 nm; the ratio of the two (I0.05) is... 487 / I 625 The concentration of sulfur dioxide derivatives exhibits a linear relationship with the concentration of sulfur dioxide derivatives within a certain range. Furthermore, it is sensitive to changes in system viscosity and can identify viscosity variations. This detection system possesses significant advantages such as a large Stokes shift (208 nm), high energy transfer efficiency (97.8%), a large spacing between the two emission peaks (138 nm), and rapid response. These suggest that this fluorescent probe can not only quantitatively detect sulfur dioxide derivatives in solution systems but also be used for imaging sulfur dioxide derivatives and viscosity within living cells; it holds promise for playing a role in clinical medical diagnosis and treatment and has broad application prospects. Attached Figure Description

[0020] Figure 1 The structure and viscosity recognition mechanism of the fluorescent probe CIJ and its reaction product with sulfur dioxide derivatives described in this invention.

[0021] Figure 2 The fluorescent probe CIJ described in this invention is effective against various analytes (e.g., F). - ,Cl - ,Br - ,I - Cys, Hcy, GSH, HS - H2O2, HClO, CH3CO 2- SO4 2- S2O3 2- NO3 - NO2 - CO3 2- SCN - Mg 2+ Ca 2+ Selectivity and anti-interference properties of responses (etc.).

[0022] Figure 3 The titration diagram (a) of the fluorescent probe CIJ described in this invention and the linear relationship between the ratio of fluorescence intensity change at 487 nm and 625 nm and the concentration of sulfur dioxide derivative (b) are shown.

[0023] Figure 4 This is a confocal fluorescence imaging image of the fluorescent probe CIJ (5μM) described in this invention on endogenous sulfur dioxide derivatives in HepG2 cells, with an excitation wavelength of 405nm, blue channel (420-600nm) and red channel (600-700nm).

[0024] Wherein: (a) is a cell imaging image under the conditions shown; (b) is a bar chart of the ratio of blue channel fluorescence intensity to red channel fluorescence intensity under the conditions corresponding to (a).

[0025] Figure 5 This is a confocal fluorescence imaging image of the fluorescent probe CIJ (5μM) described in this invention on endogenous and exogenous sulfur dioxide derivatives in LO-2 cells, with an excitation wavelength of 405nm, blue channel (420-600nm) and red channel (600-700nm).

[0026] Wherein: (a) is a cell imaging image under the conditions shown; (b) is a bar chart of the ratio of blue channel fluorescence intensity to red channel fluorescence intensity under the conditions corresponding to (a).

[0027] Figure 6This is a confocal fluorescence imaging image of HeLa cells with different viscosities using the fluorescent probe CIJ described in this invention. The excitation wavelength is 405 nm, and the red channel is 600-700 nm.

[0028] Wherein: (a) is a cell imaging image under the conditions shown; (b) is a bar chart of relative fluorescence intensity of the red channel under the conditions corresponding to (a). Detailed Implementation

[0029] The present invention will now be described in detail with reference to specific accompanying drawings and embodiments. The examples described below are merely preferred embodiments of the present invention. It should be noted that the following description is only for explaining the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, and alterations made to the embodiments based on the technical essence of the present invention shall fall within the scope of the technical solution of the present invention.

[0030] Unless otherwise specified, all materials and reagents used in the following embodiments are commercially available.

[0031] Example 1

[0032] 5-Carboxy-1,2,3,3-Tetramethyl-3H-indole-1-iodide (1.3807 g, 4 mmol) and 2,3,6,7-tetrahydro-1H,5H-pyridin[3,2,1-ij]quinoline-9-carboxaldehyde (0.8856 g, 4.4 mmol) were refluxed in anhydrous ethanol (70 mL) for 6 hours. The reaction mixture was cooled and filtered to give compound (E)-5-carboxy-1,3,3-trimethyl-2-(2-(2,3,6,7-tetrahydro-1H,5H-pyridin[3,2,1-ij]quinoline-9-yl)vinyl)-3H-indole-1-iodide (2.046 g, 96%). The intermediate (1.0568 g, 2 mmol), 7-(diethylamino)-3-(piperazin-1-carbonyl)-2H-chroman-2-one (0.7247 g, 2.2 mmol), and (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.4217 g, 2.2 mmol) were dissolved in dichloromethane (30 mL) and reacted at room temperature with stirring for 8 hours. Separation was achieved by silica gel column chromatography (DCM). (MeOH = 20:1) yielded 0.755 g of (E)-5-(4-(7-(diethylamino)-2-oxo-2H-chroman-3-carbonyl)piperazine-1-carbonyl)-1,3,3-trimethyl-2-(2-(2,3,6,7-tetrahydro-1H,5H-pyridin[3,2,1-ij]quinoline-9-yl)vinyl)-3H-indole-3-iodide (probe CIJ of this invention), with a yield of 45%.

[0033] mp = 200-202℃.

[0034] Structural confirmation spectral data:

[0035] IR:3430.47,2970.03,2926.39,2854..17,1709.84,1618.06,1514.98,1458.56,1268.21,1216.30,1117.74cm -1 .

[0036] 1 H NMR(400MHz,DMSO)δ8.16(d,J=15.2Hz,1H),8.00(s,1H),7.83(s,1H),7.70(s,2H),7.63(d, J=8.3Hz,1H),7.58(d,J=8.1Hz,1H),7.51(d,J=8.9Hz,1H),7.05(d,J=15.2Hz,1H),6.75(dd ,J=8.9,1.8Hz,1H),6.55(s,1H),3.86(s,3H),3.67(s,4H),3.45(d,J=6.1Hz,8H),3.09(q,J =7.2Hz,4H),2.74(t,J=5.7Hz,4H),1.97-1.84(m,4H),,1.72(s,6H),1.17(t,J=7.3Hz,6H). 13 C NMR (101MHz, DMSO) δ177.99,168.63,164.34,158.47,156.65,153.73,151.30,149.78,144.10,143.24,142.02,133.33,130.17,127.95,121 .91,,121.72,115.66,112.45,109.43,107.13,102.83,96.27,64.89, 50..09,45.76,44.17,32.49,26.77,26.49,20.40,15.15,12.28,8.66.

[0037] HRMS(m / z):[MI] + calcd for C 44 H 50 N5O4:712.3857, found:712.3854.

[0038] The preparation reaction of the above fluorescent probe CIJ is shown in the following formula:

[0039]

[0040] Example 2

[0041] Prepare a test solution (HEPES buffer (10 mM, pH = 7.4) for detecting sulfur dioxide derivatives in cells, using the dual-response ratio fluorescent probe CIJ described in this invention for detecting sulfur dioxide derivatives and viscosity in an organic solvent-free system.

[0042] To a 10 mL volumetric flask containing 5 μM of the probe, add 50 μM of S using a microsyringe. 2- ,HS - Cys, Hcy, GSH, CO3 2- NO3 - S2O3 2- CH3COO - SO4 2- ,Cl - ,Br - ,I - H2O2, ClO - ,Zn 2+ Ca 2+ ,K + Mg 2+ Na + Fluorescence testing was performed 5 minutes after the reaction.

[0043] The results show that the probe CIJ described in this invention has a good response and selectivity only to sulfur dioxide derivatives, and exhibits good anti-interference ability against other analytes. Figure 2 .

[0044] Example 3

[0045] Add 0-50 μM of sulfur dioxide derivatives to a 10 mL volumetric flask containing 5 μM of the fluorescent probe CIJ described in this invention using a microsyringe, and perform fluorescence testing after 5 minutes of incubation.

[0046] The results show that, with increasing concentration of sulfur dioxide derivative, the fluorescence intensity of the fluorescent probe described in this invention gradually increases at 487 nm and gradually decreases at 625 nm; the ratio of fluorescence intensity at 487 nm to fluorescence intensity at 625 nm shows a linear relationship with the concentration of sulfur dioxide derivative within a certain range. Figure 3 It can be confirmed that the fluorescent probe CIJ described in this invention can quantitatively detect low concentrations of sulfur dioxide derivatives.

[0047] Example 4

[0048] Intracellular fluorescence imaging assay

[0049] The laser scanning confocal microscope was set with an excitation wavelength of 405 nm, a blue channel collection wavelength of 420-600 nm, and a red channel collection wavelength of 600-700 nm.

[0050] After HepG2 and L-O2 cells were transferred to small glass bottles and incubated for 24 hours, they were divided into groups for experiments.

[0051] Control group: Cells were incubated with the fluorescent probe CIJ (5 μM) solution described in this invention for 1 hour, then washed three times with PBS, and imaged using a laser scanning confocal microscope. It can be seen that the cells in the control group showed stronger fluorescence in the red channel and weaker fluorescence in the blue channel.

[0052] Experimental group: Cells were first incubated with 500 μM GSH and 250 μM Na2S2O3 for 1 hour, and then incubated for another hour with 5 μM of the fluorescent probe CIJ solution described in this invention; imaging was performed using a laser scanning confocal microscope. It can be seen that the red channel fluorescence in this group was weaker than that in the control group, while the blue channel fluorescence was significantly stronger; the ratio of the statistical values ​​of the blue channel fluorescence intensity to the red channel fluorescence intensity changed very significantly. If Na2S2O3 was not added or the inhibitor TNBS was added, no endogenous SO2 derivatives were generated, and the fluorescence changes were similar to those in the control group, see [reference needed]. Figure 4 .

[0053] Alternatively, in the experimental group: L-O2 cells were first incubated with 5 μM of the fluorescent probe CIJ solution described in this invention for 1 hour, and then cultured with GSH / Na2S2O3 or different concentrations of NaHSO3 (100 μM and 500 μM) for 1 hour; imaging was performed using a laser scanning confocal microscope. It can be seen that the experimental group with GSH / Na2S2O3 showed stronger red channel fluorescence and weaker blue channel fluorescence, with no significant difference between the two and the control group; the cells with NaHSO3 showed weaker red channel fluorescence and stronger blue channel fluorescence; the ratio of the statistical values ​​of blue channel fluorescence to red channel fluorescence intensity changed significantly, see... Figure 5 .

[0054] Example 5: Fluorescence imaging test of changes in cell viscosity.

[0055] HeLa cells were incubated with nystatin (10 μM) for half an hour, followed by incubation with the fluorescent probe CIJ (5 μM) described in this invention for 1 hour. Confocal fluorescence imaging was then performed. The results showed that the fluorescence of the red channel increased with increasing viscosity. Figure 6 .

Claims

1. A dual-responsive ratiometric fluorescent probe for the detection of sulfur dioxide derivatives and viscosity in an organic solvent-free system, characterized by: The ratiometric fluorescent probe consists of an energy donor coumarin derivative fluorophore, a linker group piperazine, and an energy acceptor (…). E The chemical name is: (E)-5-(4-(7-(diethylamino)-2-oxo-2H-chromium-3-carbonyl)piperazine-1-carbonyl)-1,3,3-trimethyl-2-(2-(2,3,6,7-tetrahydro-1H,5H-pyridin[3,2,1-ij]quinoline-9-yl)vinyl)-3H-indole-3-iodide, composed of three parts; its chemical structure is shown in formula (I): Formula (I); The sulfur dioxide derivatives are bisulfite and sulfite.

2. Use of the ratiometric fluorescent probe for detecting sulfur dioxide derivatives and viscosity in an organic solvent-free system according to claim 1 for the preparation of a product for detecting samples containing sulfur dioxide derivatives, the sulfur dioxide derivatives being bisulfite and sulfite.

Images