Msrs fluorescent probe and preparation method and application thereof

Abstract

The application discloses a fluorescent probe for detecting methionine sulfoxide reductase (Msrs), a preparation method of the fluorescent probe and application of the fluorescent probe in cell imaging and biosensing. The application also discloses the preparation method of the Msrs fluorescent probe, which comprises the following steps: first, synthesizing a benzocoumarin derivative fluorophore, and then introducing a pyridine ethylene-phenyl sulfoxide unit into the fluorophore through a flexible alkyl chain to construct a novel two-photon ratio type fluorescent probe. When Msrs exists in a solution, the fluorescent probe can react with the Msrs to form a new fluorescent substance, realizes the fluorescent detection of the Msrs in a ratio mode, and further realizes the ratio type fluorescent imaging of the Msrs in cells.

Description

Technical Field

[0001] This invention belongs to the field of fluorescence imaging and biosensing technology, and relates to a fluorescent probe for detecting Msrs and its preparation method, as well as its application in cell imaging and biosensing. Background Technology

[0002] Oxidative stress is an imbalance between the production of reactive oxygen species (ROS / RNS) and antioxidant defenses in the body, potentially leading to protein and tissue damage. Generally, cells resist this oxidative damage through two pathways. One is by employing preventative mechanisms, such as superoxide dismutase and glutathione peroxidase, which can scavenge ROS before damage occurs, thus protecting the cell. The other is by repairing damaged biomolecules, including DNA and proteins. For example, methionine residues in proteins are among the most susceptible to ROS, being oxidized to methionine sulfoxide, leading to damage to protein structure and function. Methionine sulfoxide reductase specifically catalyzes the reduction of methionine sulfoxide to methionine, thereby repairing the structure of methionine-containing proteins and restoring their physiological activity. Abnormal fluctuations in methionine sulfoxide reductase (Msrs) levels are closely associated with a range of neurological disorders, such as schizophrenia, Alzheimer's disease (AD), and Parkinson's disease (PD). Given these correlations, identifying and imaging changes in Msrs levels in active neurons under oxidative stress can further elucidate the link between Msrs and these diseases.

[0003] Fluorescence sensing and imaging technologies are considered powerful tools for studying biological targets, including enzymes, due to their high sensitivity and selectivity. Most reported Msrs probes currently available detect them by detecting changes in fluorescence intensity at a single wavelength, which can be affected by probe concentration bias or environmental factors (such as the light source). Ratiometric fluorescence sensors offer a more desirable approach, relying on the ratio of fluorescence intensities emitted at two different wavelengths, thus minimizing these interferences. However, the two ratiometric fluorescent probes for detecting Msrs reported to date are designed based on the "on-off" mechanism of intramolecular charge transfer (ICT). They either exhibit highly overlapping emission peaks and lack a linear response, or require UV excitation and only show very weak ratiometric characteristics, making them unsuitable for accurate detection of Msrs in living cells. Therefore, developing novel fluorescent probes with distinct dual-channel emission, reliable ratiometric response, and long-wavelength excitation is essential. Summary of the Invention

[0004] The purpose of this invention is to provide a novel fluorescent probe for detecting Msrs and its application in cell imaging and biosensing. The fluorescent probe (shown as CPSO) has advantages such as high selectivity and good accuracy.

[0005] This invention provides a Msrs fluorescent probe with the structure shown in the formula CPSO, wherein the anion can be, but is not limited to, chloride ions (Cl...). - ), bromide ions (Br) - ), iodide ions (I) - ), hexafluorophosphate (PF6) - Any one of the following:

[0006]

[0007] The Msrs fluorescent probe is a ratiometric probe. As the Msrs concentration increases, the intensity of the original fluorescence emission peak around 432 nm gradually decreases, while the intensity of the fluorescence emission peak around 553 nm gradually increases. Quantitative analysis of Msrs can be performed by observing the change in the ratio of these two emission peak intensities. Compared with other probes, the Msrs fluorescent probe of this invention exhibits good selectivity, good biocompatibility, and quantitative analysis capabilities, making detection more accurate and providing significant advantages in biosensing and imaging.

[0008] This invention also provides a method for preparing a Msrs fluorescent probe, comprising the following steps:

[0009] Compounds 2 and 3 were reacted in acetonitrile in the first step to obtain a pale yellow solid compound CPS. Then, compound CPS and 3-chloroperoxybenzoic acid were reacted in an organic solvent in the second step to obtain the Msrs fluorescent probe CPSO. The reaction process of the preparation method is shown in the following reaction formula (a):

[0010]

[0011] The temperature of the first reaction is 80℃-90℃; preferably, it is 82℃.

[0012] The reaction time for the first step is 12h-24h; preferably, it is 24h.

[0013] Preferably, after the first step reaction is completed, the mixture is cooled to room temperature and added dropwise to ethyl acetate to obtain CPS, and then the CPS is reacted with 3-chloroperoxybenzoic acid in the second step reaction.

[0014] The temperature of the second step reaction is 0℃-25℃; preferably, it is 0℃.

[0015] The first step reaction is preferably carried out in a pressure-resistant tube.

[0016] The organic solvent is selected from one or more of dichloromethane, 1,2-dichloroethane, etc.; preferably, it is 1,2-dichloroethane.

[0017] The reaction time in the second step is 10h-18h; preferably, it is 18h.

[0018] The molar ratio of compound 2 to compound 3 is 1:1;

[0019] The molar ratio of the compound CPS to 3-chloroperoxybenzoic acid is 1:1.2.

[0020] The synthesis method of compound 2 is as follows: Compound 1, 1,3-dibromopropane, and K₂CO₃ are dissolved in an organic solvent and heated to reflux. After the reaction is complete, the mixture is extracted with distilled water and dichloromethane. The solvent is removed by vacuum distillation, and compound 2 is obtained by silica gel column chromatography (PE / EA = 3 / 1, v / v). The reaction process is shown in reaction formula (b):

[0021]

[0022] The organic solvent is selected from one or more of acetonitrile, acetone, or N,N-dimethylformamide; preferably, it is acetone.

[0023] The reaction temperature is 50℃-60℃; preferably, it is 56℃.

[0024] The reaction time is 12h-24h; preferably, it is 24h.

[0025] The molar ratio of compound 1, 1,3-dibromopropane and K2CO3 is 1:5:3.

[0026] The preparation steps of reaction (c) are as follows: compound 3 and 3-chloroperoxybenzoic acid are dissolved in an organic solvent and reacted.

[0027] After the reaction was complete, the compound was added dropwise to ethyl acetate to obtain compound 4.

[0028]

[0029] The organic solvent is selected from one or more of acetonitrile, acetone, or dichloromethane; preferably, it is dichloromethane.

[0030] The reaction temperature is 0℃-25℃; preferably, it is 0℃.

[0031] The reaction time is 12h-18h; preferably, it is 18h.

[0032] The molar ratio of compound 3 to 3-chloroperoxybenzoic acid is 1:1.

[0033] The preparation steps of reaction (d) are as follows: Compound 3 and 1,3-dibromopropane are dissolved in an organic solvent and heated under reflux. After the reaction is complete, the mixture is recrystallized from ethyl acetate to obtain a pale yellow solid compound 5.

[0034]

[0035] The organic solvent is selected from one or more of acetonitrile, acetone, or dichloromethane; preferably, it is acetonitrile.

[0036] The reaction temperature is 70℃-90℃; preferably, it is 85℃.

[0037] The reaction time is 12h-24h; preferably, it is 24h.

[0038] The molar ratio of compound 3 to 3-chloroperoxybenzoic acid is 1:10.

[0039] The preparation steps of reaction (e) are as follows: Compound 4 and 1,3-dibromopropane are dissolved in an organic solvent and heated under reflux. After the reaction is complete, the mixture is recrystallized with anhydrous diethyl ether, stirred until powder precipitates, filtered, and washed with acetone to obtain a white solid compound 6.

[0040]

[0041] The organic solvent is selected from one or more of acetonitrile, acetone, or dichloromethane; preferably, it is acetonitrile.

[0042] The reaction temperature is 70℃-90℃; preferably, it is 85℃.

[0043] The reaction time is 12h-24h; preferably, it is 12h.

[0044] The molar ratio of compound 4 to 1,3-dibromopropane is 1:10.

[0045] This invention also proposes a method for constructing a Msrs fluorescent probe. First, a fluorophore of compound 2 (benzocoumarin derivative) is synthesized. Then, a pyridine-ethylene-phenyl sulfoxide unit of compound 4 is introduced into the fluorophore via a flexible alkyl chain to construct a novel two-photon ratiometric fluorescent probe. When Msrs are present in solution, the pyridine-ethylene-phenyl sulfoxide unit in the fluorescent probe is catalytically reduced by Msrs to a new fluorescent substance, pyridine-phenyl sulfide unit (compound 5), forming a new fluorescence emission peak. This enables ratiometric fluorescence detection of Msrs and further allows for ratiometric fluorescence imaging of Msrs within cells.

[0046] In one specific embodiment of the present invention, the method for preparing the Msrs fluorescent probe includes:

[0047] Compounds 2 (63.5 mg, 0.28 mmol) and 3 (93.3 mg, 0.28 mmol) were dissolved in 6 mL of acetonitrile and refluxed for 24 hours. After cooling to room temperature, the solution was added dropwise to ethyl acetate, precipitating a pale yellow product, namely compound CPS, which was obtained by filtration. Then, CPS (50 mg, 0.09 mmol) and 3-chloroperoxybenzoic acid (85%, 15.5 mg, 0.09 mmol) were dissolved in 6 mL of dichloromethane and refluxed for 24 hours. After cooling to room temperature, the solution was added dropwise to ethyl acetate, precipitating a white solid product, namely the Msrs fluorescent probe CPSO, which was obtained by filtration.

[0048] The present invention also provides a Msrs fluorescent probe prepared by the method described above.

[0049] The Msrs fluorescent probe described in this invention is a ratiometric probe, a two-photon ratiometric fluorescent probe with high selectivity for Msrs. As the Msrs concentration increases, the intensity of the probe's original fluorescence emission peak around 432 nm gradually decreases, while the intensity of the fluorescence emission peak around 553 nm gradually increases. Quantitative analysis of Msrs can be performed by observing the change in the ratio of these two emission peak intensities. Compared with other probes, the Msrs fluorescent probe described in this invention has good selectivity, good biocompatibility, high stability, and quantitative analysis capabilities, making detection more accurate and offering significant advantages in biosensing and imaging.

[0050] The present invention also provides the application of the Msrs fluorescent probe in the detection of Msrs in vitro and / or intracellular and / or in vivo; the detection of Msrs is achieved by two-photon fluorescence excitation; wherein the cells include, but are not limited to, neuronal cells, HeLa cells, etc.; the cells do not include human cells; the in vivo does not include humans.

[0051] In some specific embodiments, in the fluorescence detection method, the buffer solution is Tris-HCl buffer; and / or, the reaction temperature is constant at 37°C; and / or, the reaction time is 60 min.

[0052] This invention also provides the application of the Msrs fluorescent probe in cell imaging and biosensing.

[0053] Because the Msrs fluorescent probe can react with Msrs to form a new fluorescent substance, the original fluorescence emission peak at around 432 nm is gradually quenched, and a gradually increasing fluorescence emission peak appears at 553 nm, showing a ratiometric fluorescence output. Therefore, this invention also provides an in vitro fluorescence detection method for Msrs, which includes the following steps: mixing the Msrs fluorescent probe of this invention with Msrs in a buffer solution and shaking to react; measuring the ratio change of fluorescence intensity of the system under excitation by a light source at a wavelength of 700 nm (two-photon excitation) or 405 nm (single-photon excitation), thereby achieving quantitative detection of Msrs.

[0054] The buffer solution includes, but is not limited to, Tris-HCl buffer solution; preferably, the concentration of the Tris-HCl buffer solution is 25 mM and the pH is 7.4.

[0055] The reaction temperature is 37°C.

[0056] The reaction time is 30 min to 90 min; preferably, it is 60 min.

[0057] The linear range of the method is 0.1-1 μg / mL; the limit of detection is 0.01 μg / mL.

[0058] The linear relationship between the two-photon fluorescence intensity ratio and the Msrs concentration is R. 2 =0.992.

[0059] The method can be applied to cells and living organisms.

[0060] In one specific embodiment of the present invention, the in vitro Msrs fluorescence detection method specifically includes: taking 0.575 mg of the Msrs fluorescent probe prepared by the above method, diluting it to 100 mL with a buffer containing Tris-HCl, and adding different volumes of 500 μg / mL Msrs to each 200 mL of the above solution. Then, under excitation by a light source at a wavelength of 700 nm (two-photon excitation) or 405 nm (single-photon excitation), the fluorescence intensity change of the system is measured. As the concentration of Msrs increases, the original fluorescence emission peak at approximately 432 nm of the probe gradually quenches, and a gradually increasing fluorescence emission peak appears at 553 nm, showing a ratiometric fluorescence output. It exhibits good linearity in the range of 0.1-1 μg / mL, with a detection limit of 0.01 μg / mL, which can be used for effective detection of Msrs. Figure 3 .

[0061] In one specific embodiment of the present invention, the method for intracellular Msrs fluorescence imaging includes the following steps: incubating cells with the Msrs probe, adding buffer solution, and then observing using a confocal microscope. The cells are excited with an excitation wavelength of 700 nm. As the Msrs increase, the fluorescence emission of the probe at approximately 432 nm gradually decreases, while the fluorescence at 553 nm gradually increases, resulting in a ratio response. This allows for the realization of ratio fluorescence response of intracellular Msrs and imaging analysis.

[0062] The incubation temperature is 37°C; the incubation time between the cells and the Msrs probe is 10 min to 60 min.

[0063] The beneficial effects of this invention include: First, a novel two-photon ratiometric fluorescent probe with high selectivity for Msrs (MSR) was synthesized by introducing two chlorine atoms and an aldehyde group into the fluorophore of a coumarin derivative. This probe is a molecular platform integrating intramolecular charge transfer (ICT) and fluorescence resonance energy transfer (FRET) mechanisms. By activating the FRET system between the energy donor and energy acceptor, a ratiometric probe was successfully constructed, overcoming the limitation of traditional single-detection-wavelength fluorescent probes in quantitatively detecting Msrs levels in organisms, thus improving the accuracy of Msrs detection. Simultaneously, this probe can be excited using a near-infrared (700 nm) two-photon long-wavelength excitation source, reducing photodamage to organisms and increasing tissue penetration depth, which is beneficial for detecting Msrs in living cells and tissues. Furthermore, this invention provides the first investigation into changes in Msrs in neurons under oxidative stress and fluorescence imaging of Msrs in brain slices from AD mice. Attached Figure Description

[0064] Figure 1 This represents the 1H NMR spectrum of the Msrs fluorescent probe molecule of this invention.

[0065] Figure 2 Absorption and fluorescence spectra of compounds 2 and 6 (A); absorption and fluorescence spectra of compounds 2 and 5 (B).

[0066] Figure 3 The fluorescence spectra of compounds 5 and 6 in this invention are shown.

[0067] Figure 4 The values ​​represent the two-photon absorption cross section and fluorescence lifetime of compounds CPSO and 2 in this invention.

[0068] Figure 5(A) The 1H NMR spectrum (p) of compound CPSO in this invention, showing the reaction of CPSO with MsrA for 20 min (q), 40 min (r), 60 min (s) and CPS (t) in D2O at 298 K. (B) CPSO (e), CPSO reacting with MsrA (1.5 μg / mL) at 37 °C for 1 h (f) High-performance liquid chromatography (HPLC) of CPS (g). ([CPSO] = [CPS] = 10 μM). (C) High-resolution mass spectra of the reaction products of probe CPSO (10 μM) and MsrA (1.5 μg / mL).

[0069] Figure 6 The fluorescence emission spectra (A) and the linear relationship of the ratio change (B) of the Msrs fluorescent probe of this invention in response to different concentrations of MsrA (0.1-4 μg / mL) are shown.

[0070] Figure 7 This invention relates to the Msrs fluorescent probe's time response to MsrA.

[0071] Figure 8 This diagram illustrates the selectivity and interference experiments of the Msrs fluorescent probe for Msrs detection in this invention. The bars on the left represent the individual presence of various interfering amino acids, while the bars on the right represent the coexistence of Msrs with potential interfering amino acids (A), reactive oxygen species (B), and ions (C). The bars on the left represent the selectivity experiment, and the bars on the right represent the interference experiment.

[0072] Figure 9 The present invention relates to the Msrs fluorescent probe for fluorescence imaging of different concentrations of Msrs within neuronal cells. Detailed Implementation

[0073] The present invention will be further described in detail below with reference to the specific embodiments and accompanying drawings. Except for the contents specifically mentioned below, the processes, conditions, and experimental methods for implementing the present invention are all common knowledge and general knowledge in the art, and the present invention does not have any particular limitations.

[0074] Example 1. Preparation of Msrs fluorescent probe

[0075] Compound 2 (63.5 mg, 0.28 mmol) and compound 3 (93.3 mg, 0.28 mmol) mentioned in the previous text were dissolved in 6 mL of acetonitrile and refluxed for 24 hours. After cooling to room temperature, the solution was added dropwise to ethyl acetate, and a precipitate was formed. Filtering yielded a pale yellow product, namely compound CPS. Then, CPS (50 mg, 0.09 mmol) and 3-chloroperoxybenzoic acid (85%, 15.5 mg, 0.09 mmol) were dissolved in 6 mL of dichloromethane and refluxed for 24 hours. After cooling to room temperature, the solution was added dropwise to ethyl acetate, and a precipitate was formed. Filtering yielded a white solid product, namely the Msrs fluorescent probe CPSO (yield 65%). 1 HNMR(500MHz,MeOD)δ:8.00(s,3H),7.94(d,J=7.7Hz,3H),7.55(d,J=7.9Hz,3H),7.44(t,J=7.8Hz,3H),7.23(d,J=2.3Hz,2 H),7.02–6.98(m,1H),6.97(s,1H),6.64(s,1H),5.06–4.94(m,2H),4.05(t,J=6.6Hz,2H),2.18(s,3H),1.64–1.55(m,2H). 13 CNMR (126MHz, MeOD) δ: 162.05, 160.15, 157.81, 155.22, 143.92, 143.38, 141.59, 140.18, 133.35, 129.59, 128.79, 12 8.63, 127.59, 127.28, 125.48, 125.29, 118.65, 118.25, 112.51, 111.20, 107.41, 96.10, 65.98, 58.87, 29.94, 20.58. HRMS(ESI):C 30 H 26 NO4S[M-Br]+ calcd 496.1577, found: 496.1569. See NMR image. Figure 1 .

[0076] Example 2. Fluorescence resonance energy transfer (FRET) mechanism before and after the reaction of probe CPSO with Msrs

[0077] Using Tris-HCl buffer (25 mM, pH = 7.4) as the solvent, 10 μM solutions of compounds 2, 5, and 6 mentioned in the preceding text were prepared, and their absorption and fluorescence spectra were tested (e.g., ...). Figure 2The fluorescence spectrum of compound 2 showed a very significant spectral overlap with the absorption spectrum of compound 5, while the spectral overlap between compound 2 and compound 6 was very small. This clearly confirms the feasibility of the Msrs-catalyzed reduction of FRET activation process.

[0078] Example 3. Intramolecular charge transfer (ICT) mechanism before and after the reaction of probe CPSO with Msrs

[0079] Using Tris-HCl buffer (25 mM, pH = 7.4) as the solvent, 10 μM solutions of compounds 5 and 6, as mentioned in the preceding text, were prepared, and their fluorescence spectra were tested. Figure 3 As shown, the initial compound 6 does not emit fluorescence due to the electron-withdrawing properties at both ends of its structure. However, when the sulfoxide group is converted to a thioether (compound 5), the fluorescence intensity is significantly enhanced, increasing by approximately 17-fold. This is due to the significant electron-donating effect in the conjugated structure of compound 5. Therefore, the feasibility of dual regulation of redox-dependent FRET and ICT is confirmed through the structural transformation between sulfide / sulfoxide compounds 5 and 6.

[0080] Example 4. Two-photon performance and FRET efficiency of probe CPSO

[0081] Next, the two-photon absorption cross section of the CPSO probe prepared in Example 1 of this invention was determined using a reference method. The first step was to calculate the fluorescence quantum yield of the probe, using the following formula:

[0082] Φ s =Φ r (A r F s n s 2 ) / (A s F r n r 2 )

[0083] s and r represent the sample and standard sample, respectively. Let A be the fluorescence quantum yield, F be the absorbance of the molecule, F be the overall emission area, and n be the refractive index. Next, using a reference material to compare fluorescence measurements, the two-photon cross-section is determined according to the following formula:

[0084] σ s =σ r (F s Φ r C r n r ) / (F r Φ r C s n s )

[0085] The subscripts s and r represent the sample and reference sample, σ is the two-photon cross section, and F is the measured two-photon fluorescence intensity. Let C be the fluorescence quantum yield, C be the solution concentration, and n be the refractive index. For the measurement of the two-photon cross-section of the probe CPSO, Rhodamine 6G was chosen as a reference, as its fluorescence maximum value is close to that of the probe. Finally, the two-photon interaction cross-section is obtained by the following equation:

[0086] σ′=Φ×σ

[0087] φ is the fluorescence quantum yield, and σ is the two-photon cross-section. For example... Figure 4 As shown in Figure A, after comprehensively considering the near-infrared region and the two-photon absorption cross-section of TPA, 700 nm was selected as the excitation wavelength for subsequent experiments. Then, under the same conditions, the fluorescence lifetimes of compound 2, CPSO, and CPS were measured (e.g., ...). Figure 4 (B) was used to evaluate the FRET efficiency of CPSO and CPS. Donor compound 2 had the longest lifetime at 5.15 ns, while the probe CPSO had a slightly shorter lifetime of 4.37 ns. The lifetime decrease was particularly pronounced in CPS (1.25 ns), clearly indicating that CPS has a very high FRET efficiency, calculated at 76%, compared to only 15% in CPSO.

[0088] Example 5. Reaction process of probe CPSO and MsrA

[0089] Using Tris-HCl buffer (25 mM, pH = 7.4) as a solvent, 10 μM of the CPSO and CPS prepared in Example 1 of this invention were used for analysis and detection by nuclear magnetic resonance titration, high performance liquid chromatography, and high resolution mass spectrometry. Figure 5 As shown, the NMR of the mixture gradually changes, with the hydrogen at the methyl position shifting to a lower field. This is due to the electron-donating effect generated after the sulfur-oxygen double bond is reduced to a methyl-thio bond. Figure 5 (A) Then, high-performance liquid chromatography (HPLC) was used to further track the conversion process of MsrA-catalyzed reduction of CPSO. For example... Figure 5 As shown in B, the reaction mixture of the probe and MsrA exhibits two substances with excellent retention times. Figure 5 The B curve f in the figure, and the corresponding reference sample ( Figure 5 Comparing curves B (e and g) in the data, the two substances are identified as the probe CPSO and the reduction product CPS, respectively. Furthermore, high-resolution mass spectrometry (HR-MS) analysis further confirmed that MsrA catalyzes the conversion of CPSO to CPS. Figure 5In the C), in addition to the [CPSO-Br]+ peak, a new peak with an m / z value of 480.1608 ([CPS-Br]+, referred to as C30H26NO3S+) also appeared, which corresponds to the previously speculated enzymatic hydrolysis product CPS.

[0090] Example 6. Detection of Msrs in vitro

[0091] (1) Preparation of fluorescence intensity calibration curve

[0092] Take 0.575 mg of the Msrs fluorescent probe prepared by the above method, dilute it to 100 mL with Tris-HCl buffer, and add different volumes of 500 μg / mL Msrs to each of the above solutions in 200 mL. Then measure the fluorescence intensity of the solution at an excitation wavelength of 700 nm. As the concentration of Msrs added increases, the fluorescence intensity of the probe itself gradually decreases, and the intensity of the new fluorescence emission peak gradually increases, showing good linearity in the range of 0.1-1 μg / mL. The limit of detection is 0.01 μg / mL, and the linear relationship is R. 2 =0.992. See Figure 5 .

[0093] (2) In vitro sample detection

[0094] Take 0.575 mg of the Msrs fluorescent probe prepared by the above method, dilute it to 100 mL with Tris-HCl buffer, and take 200 mL of the above solution to add different volumes of Msrs at a concentration of 500 μg / mL. Then measure the fluorescence intensity of the solution at an excitation wavelength of 700 nm. As the concentration of Msrs added increases, the fluorescence intensity of the probe itself gradually decreases, and the intensity of the new fluorescence emission peak gradually increases, showing good linearity in the range of 0.1-1 μg / mL. The limit of detection is 0.01 μg / mL, indicating that the Msrs fluorescent probe of the present invention can be used for effective detection of Msrs.

[0095] Example 7. Reaction kinetics of probe CPSO and Msrs

[0096] Using Tris-HCl buffer (25 mM, pH = 7.4) as a solvent, 10 μM of the CPSO prepared in Example 1 of this invention was mixed with different concentrations of MsrA, and the dynamic response of the probe to MsrA was measured. Figure 7 As shown, the response time of CPSO to MsrA gradually increases with increasing MsrA concentration. When the MsrA concentration reaches 4 μg / mL, the reaction time between the probe and MsrA reaches its fastest, approximately 8 minutes, which is much faster than that of previously reported MsrA probes. This is attributed to the high specificity and ratiometric fluorescence sensing mode of the CPSO probe.

[0097] Example 8. Selectivity and anti-interference ability of the Msrs fluorescent probe prepared in Example 1

[0098] To evaluate the selectivity and anti-interference ability of the Msrs fluorescent probe prepared in Example 1 of this invention, the effects of Cys, GSH, AA, Glu, BSA, DTT, Thr, His, Gly, Hcy, and metal ion K were investigated. + Na + Cu 2+ Zn 2+ Ca 2+ Mg 2+ Cu + Fe 3+ Fe 2+ Co 2+ Ni 2+ Al 3+ ; and common reactive oxygen species / reactive nitrogen species SO2, H2S, ·OH, O2 ·一 ONOO 一 HClO, NO, HNO, ROO 一 , 1 Changes in fluorescence signal when O2 is present alone and when it coexists with Msrs.

[0099] Depend on Figure 8 It is known that common amino acids, metal ions, and reactive oxygen species / reactive nitrogen species have no significant effect on Msrs detection (interference <10%). The selectivity and anti-interference experiments show that the Msrs fluorescent probe in this invention has good selectivity and anti-interference ability.

[0100] Example 9. Fluorescence imaging of intracellular Msrs

[0101] The Msrs fluorescent probe prepared in Example 1 of this invention was used for confocal fluorescence imaging of Msrs in chromaffin cells. Cultured neurons were incubated with the Msrs fluorescent probe for 1 hour, then the cell culture medium was aspirated and PBS buffer was added. Cell morphology was then determined using a confocal microscope, and fluorescence imaging was performed using fluorescence excitation at a wavelength of 700 nm. As Msrs levels increased, the fluorescence of the probe in the cell gradually decreased, while another new fluorescence gradually increased. This demonstrates that the Msrs fluorescent probe prepared in Example 1 of this invention can be used for intracellular Msrs fluorescence imaging. Two-photon fluorescence has the advantage of strong penetration, and ratiometric mode detection of Msrs can reduce interference from environmental factors, achieving accurate quantitative detection of Msrs within cells.

[0102] The scope of protection of this invention is not limited to the above embodiments. Any variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

Claims

1. A Msrs fluorescent probe, characterized in that, Its structure is shown in the formula CPSO, where the anion is a bromide ion Br. - :

2. A method for preparing the Msrs fluorescent probe as described in claim 1, characterized in that, The method is as follows: first, the fluorophore of compound 2 benzocoumarin derivative is synthesized, and then the Msrs fluorescent probe CPSO is constructed by introducing compound 4 pyridine vinyl-phenyl sulfoxide unit into the fluorophore through a flexible alkyl chain; The method includes the following steps: reacting compound 2 and compound 3 in acetonitrile to obtain a pale yellow solid compound CPS; then reacting compound CPS and 3-chloroperoxybenzoic acid in an organic solvent to obtain the Msrs fluorescent probe CPSO; the reaction process is shown in the following reaction formula (a): The flexible alkyl chain refers to -CH2CH2CH2-; The structure of compound 4 is as follows:

3. The method as described in claim 2, characterized in that, The temperature of the first reaction is 82°C; And / or, the duration of the first reaction is 12h-24h; And / or, the temperature of the second reaction is 0°C-25°C; And / or, the second reaction takes 10-18 hours; And / or, the molar ratio of compound 2 and compound 3 is 1:1; And / or, the molar ratio of the compound CPS to 3-chloroperoxybenzoic acid is 1:1.

2.

4. The method as described in claim 2, characterized in that, The synthesis method of compound 2 is as follows: compound 1 is dissolved in an organic solvent with 1,3-dibromopropane and K2CO3, heated to reflux, and after the reaction is completed, it is extracted with distilled water and dichloromethane, the solvent is removed by vacuum distillation, and the product is obtained by silica gel column chromatography. The reaction process is as follows:

5. The method as described in claim 4, characterized in that, The organic solvent is selected from one or more of acetonitrile, acetone or N,N-dimethylformamide; and / or, the reaction temperature is 50℃-60℃; and / or, the reaction time is 12h-24h; and / or, the molar ratio of compound 1, 1,3-dibromopropane and K2CO3 is 1:5:

3.

6. The Msrs fluorescent probe as described in claim 1, characterized in that, It is a two-photon ratiometric fluorescent probe with high selectivity for Msrs.

7. The application of the Msrs fluorescent probe as described in claim 1 in in vitro and / or intracellular detection of Msrs not for the purpose of disease diagnosis, and in cell imaging and biosensing.

8. The application as described in claim 7, characterized in that, The cells mentioned do not include those derived from humans.

9. A method for detecting Msrs fluorescence in vitro and / or intracellularly not for the purpose of disease diagnosis, characterized in that, The method includes the following steps: reacting the Msrs fluorescent probe as described in claim 1 with Msrs in a buffer solution, then exciting with an excitation wavelength of 700 nm, and measuring the fluorescence intensity of the solution. As the content of added Msrs increases, the fluorescence intensity of the Msrs fluorescent probe itself gradually decreases, and the fluorescence intensity of a new emission peak gradually increases, showing a ratio-type change with good linearity, thereby achieving quantitative detection of Msrs.

10. The fluorescence detection method as described in claim 9, characterized in that, The buffer solution is Tris-HCl buffer; and / or, the reaction temperature is constant at 37°C; and / or, the reaction time is 60 min.

11. A method for intracellular Msrs fluorescence imaging not intended for disease diagnosis, characterized in that, The method includes the following steps: co-incubating cells with the Msrs fluorescent probe as described in claim 1, then adding buffer solution, and then observing using a confocal microscope, using a pulsed laser with an excitation wavelength of 700 nm to excite the cells and observe the fluorescence emission of the cells at 432 nm; as the concentration of Msrs increases, the fluorescence emission of the probe in the cells at 432 nm gradually decreases, and the fluorescence at 553 nm gradually increases, resulting in a ratiometric response, thereby realizing the ratiometric fluorescence response of Msrs in the cells and performing imaging analysis.

12. The method as described in claim 11, characterized in that, The incubation temperature is 37°C; and / or the incubation time of the cells with the Msrs probe is 10 min to 60 min.

Images