Fluorescent probe for recognizing methylglyoxal as well as preparation method and application of fluorescent probe

Abstract

The invention discloses a fluorescent probe for recognizing methylglyoxal as well as a preparation method and application of the fluorescent probe, and belongs to the technical field of chemical analysis and detection. The structural formula of the fluorescent probe for identifying methylglyoxal is shown in the specification. In addition, the invention also provides a preparation method of the fluorescent probe. In addition, the invention also provides an application of the fluorescent probe or the fluorescent probe prepared by the preparation method in detection of methylglyoxal in an environment or a biological sample. The fluorescent probe provided by the invention can be used for detecting methylglyoxal in living cells and zebra fish, and can realize distribution imaging and detection of MGO in an arabidopsis thaliana living body in a lossless manner.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of chemical analysis detection, and particularly relates to a fluorescent probe for recognizing methylglyoxal and a preparation method and application thereof. BACKGROUND

[0002] Methylglyoxal (MGO) is an active alpha, beta-dicarbonyl ketone aldehyde, which is mainly produced in the physiological system through metabolic pathways such as glucose, alcohol and protein. Methylglyoxal is the product of spontaneous conversion of phosphotriose acid and is an endogenous dicarbonyl metabolite, which plays a key role in plant growth and development, signal transduction and non-stress response. However, methylglyoxal has a dual role in plants. High concentration of methylglyoxal can have obvious toxic effects on plant cells, such as inhibiting seed germination, hindering normal development of root system and interfering with photosynthesis. Obviously, the concentration of methylglyoxal in plants plays a key role in ensuring the optimal function of physiological processes. In addition, the concentration of methylglyoxal is closely related to the ability of plants to resist stress, such as drought, salinity and extreme temperature. Therefore, methylglyoxal is considered as a potential biochemical marker for evaluating the ability of plants to resist stress. In summary, the detection of methylglyoxal in plants is of great significance for further studying the growth and development mechanism and stress response of plants.

[0003] Fluorescence imaging technology can provide more intuitive and accurate data support, and thus becomes a powerful tool for studying the distribution of methylglyoxal in vivo and its interaction with other metabolites. So far, several fluorescent probes for recognizing methylglyoxal have been developed, and almost all of these probes are developed for animal and cell research, and few probes are used to study the influence of methylglyoxal content on plant growth process, and these probes are usually applied to biological fluorescence microscope imaging. Compared with the limitation of observing part of plant tissue by fluorescence microscope imaging, the use of in vivo fluorescence imaging instrument can image under the premise that the plant is alive and the tissue structure is not damaged, and can present the distribution rule of methylglyoxal at the level of plant organs and even whole plants, reflecting the correlation between local changes and overall physiological state. Due to the influence of plant structure and external environment, in vivo fluorescence imaging is more difficult than microscope fluorescence imaging, and therefore the probe needs to have better light stability, stronger tissue penetration ability and stronger environmental tolerance. Therefore, there is an urgent need to develop a new fluorescent probe which can be used for in situ in vivo imaging of animals and plants. SUMMARY

[0004] The present application aims to overcome the above technical deficiencies, and provides a fluorescent probe for recognizing methylglyoxal and a preparation method and application thereof, which solves the technical problem of how to realize in situ in vivo imaging of animals and plants in the prior art.

[0005] To achieve the above technical purpose, the technical scheme of the present application provides a fluorescent probe for identifying methylglyoxal, which has a structural formula as follows: (I).

[0006] In any embodiment, the fluorescent probe has a maximum emission wavelength at 530 nm after responding to the product of methylglyoxal, and has green fluorescence.

[0007] In any embodiment, the product has a structural formula as follows: (II).

[0008] In addition, the present application further provides a preparation method of the fluorescent probe, which comprises the following steps: S1, mixing concentrated nitric acid and concentrated sulfuric acid to obtain a mixed solution, then mixing and reacting p-acetamidobenzaldehyde and the mixed solution, and then separating and purifying to obtain an intermediate A-2; dissolving 3-hydroxy-3-methyl-2-butanone in anhydrous ethanol, adding malononitrile and potassium carbonate, and then heating and reacting, and then separating and purifying to obtain an intermediate A-4; The intermediate A-2 has a structural formula as follows: ; The intermediate A-4 has a structural formula as follows: ; S2, dissolving the intermediate A-2 and the intermediate A-4 in anhydrous acetonitrile, adding piperidine, and then refluxing and reacting, and then separating and purifying to obtain an intermediate A-5; The intermediate A-5 has a structural formula as follows: ; S3, dissolving the intermediate A-5 in ethyl acetate, adding concentrated hydrochloric acid, and then refluxing and reacting, and then separating and purifying to obtain an intermediate A-6; The intermediate A-6 has a structural formula as follows: ; S4, dissolving the compound A-6 in ethyl acetate, adding stannous chloride dihydrate and concentrated hydrochloric acid, and then heating and reacting, and then separating and purifying to obtain the fluorescent probe.

[0009] In any embodiment, in step S1, the mixing and reacting time is 1-1.5 h; and / or, in step S2, the refluxing and reacting time is 1-3 h; and / or, in step S3, the refluxing and reacting time is 22-25 h; and / or, in step S4, the heating and reacting time is 10-24 h.

[0010] In any embodiment, in step S1, the heating and reacting temperature is 75-85℃.

[0011] In any embodiment, in step S2 or step S3, the temperature of the reflux reaction is 75-85℃.

[0012] In any embodiment, in step S4, the temperature of the heating reaction is 65-75℃.

[0013] In addition, the application also provides a use of the fluorescent probe or the fluorescent probe prepared by the preparation method in detecting methylglyoxal in an environment or a biological sample.

[0014] In any embodiment, the use comprises imaging detection of cells, zebrafish or Arabidopsis thaliana, and the cells are suitable for non-therapeutic purposes.

[0015] Compared with the prior art, the application has the following beneficial effects: the fluorescent probe prepared by the application has low fluorescent background, good chemical stability, high sensitivity (0.14 μM) and high selectivity; the fluorescent probe can not only detect methylglyoxal in living cells and zebrafish, but also realize distribution imaging and detection of MGO in vivo of Arabidopsis thaliana without damage. The fluorescent probe prepared by the application has a large Stokes shift (120 nm) and strong anti-interference ability, and can specifically detect methylglyoxal. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 The fluorescent probe obtained in Example 1 of the application is subjected to H NMR spectrum. 1 H NMR spectrum.

[0017] Figure 2 The fluorescent probe obtained in Example 1 of the application is subjected to C NMR. 13 C NMR.

[0018] Figure 3 The fluorescent probe obtained in Example 1 of the application is subjected to HRMS spectrum.

[0019] Figure 4 The fluorescent probe obtained in Example 2 of the application is subjected to fluorescence spectrum for detecting methylglyoxal response.

[0020] Figure 5 The fluorescent probe obtained in Example 3 of the application is subjected to fluorescence spectrum for reacting with different concentrations of methylglyoxal.

[0021] Figure 6 The fluorescent probe obtained in Example 4 of the application is subjected to limit of detection test diagram for detecting methylglyoxal in a low concentration range.

[0022] Figure 7 The fluorescent probe obtained in Example 5 of the application is subjected to fluorescence response column chart for reacting with different analytes.

[0023] Figure 8A fluorescence imaging map of the fluorescent probe of the present application embodiment 6 for detecting methylglyoxal in living cells.

[0024] Figure 9 A fluorescence imaging map of the fluorescent probe of the present application embodiment 7 for detecting methylglyoxal in living zebrafish.

[0025] Figure 10 A fluorescence imaging map of the fluorescent probe of the present application embodiment 8 for detecting methylglyoxal in Arabidopsis root tip tissue.

[0026] Figure 11 A fluorescence imaging map of the fluorescent probe of the present application embodiment 9 for detecting the distribution of MGO in situ in living Arabidopsis. DETAILED DESCRIPTION

[0027] The ranges disclosed herein are defined by their lower and upper limits. Ranges can be defined by any upper and lower limits, whether or not they are expressly disclosed. Ranges include any and all intermediate ranges, whether or not explicitly stated. For example, a range of "about 0.1 to 10" is intended to include any and all intermediate ranges, e.g., "about 1 to 5," "about 0.5 to 2," "about 0.1 to 4," etc. The same applies to ranges expressed in terms of minimum and maximum values. For example, a range of "about 1 to about 10" is intended to include any and all intermediate ranges, e.g., "about 2 to about 8," "about 3 to about 7," "about 4 to about 6," etc. In other words, unless expressly stated otherwise, any range of values (of a parameter) disclosed herein is intended to include any and all sub-ranges of values within the range. For example, a range of "0 to 10" is intended to include any and all sub-ranges between (and including) the minimum value of zero and the maximum value of ten, e.g., 0 to 4.9, 0.1 to 4, 3.5 to 10, 100 to 10, etc. In addition, it is specifically intended that expressions such as "between A and B" include the values of A and B. For example, the expression "between 1 and 10" is intended to include the values of 1 and 10. Unless expressly stated otherwise, the term "about" preceding a value means that the value can vary from the stated value by as much as 10%.

[0028] Unless otherwise indicated, the terms "including", "includes" and "include" are intended to be open-ended and also to permit inclusion of other components, steps, features, etc. unless expressly stated otherwise.

[0029] Unless otherwise indicated, the term "or" is intended to be inclusive in this application, meaning A or B or both A and B. More specifically, the phrase "A or B" means any of the following: A, B, or both A and B. In other words, when analyzing a phrase that uses "or" to join a list of elements, the analysis can be done as though "or" was the exclusive or, unless otherwise stated. For example, if a phrase states "A or B," then the phrase can be interpreted as meaning "A, B, or both A and B." In other words, any of the following conditions can satisfy the condition "A or B": A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or A and B are both true (or exist).

[0030] The embodiment provides a fluorescent probe for identifying methylglyoxal, and a structural formula of the fluorescent probe is as follows: (I).

[0031] In some embodiments, the fluorescent probe has a maximum emission wavelength at 530 nm and green fluorescence after responding to the product of methylglyoxal, and a structural formula of the product is as follows: (II).

[0032] The embodiment further provides a preparation method of the fluorescent probe, and the preparation method comprises the following steps: S1, mixing concentrated nitric acid and concentrated sulfuric acid to obtain a mixed solution, mixing p-acetamidobenzaldehyde and the mixed solution and reacting for 1-1.5 h, and then separating and purifying to obtain an intermediate A-2; dissolving 3-hydroxy-3-methyl-2-butanone in anhydrous ethanol, adding malononitrile and potassium carbonate, and heating and reacting at 75-85 °C, and then separating and purifying to obtain an intermediate A-4; A structural formula of the intermediate A-2 is as follows: A structural formula of the intermediate A-4 is as follows: S2, dissolving the intermediate A-2 and the intermediate A-4 in anhydrous acetonitrile, adding piperidine, and refluxing and reacting at 75-85 °C for 1-3 h, and then separating and purifying to obtain an intermediate A-5; A structural formula of the intermediate A-5 is as follows: S3, dissolving the intermediate A-5 in ethyl acetate, adding concentrated hydrochloric acid, and refluxing and reacting at 75-85 °C for 22-25 h, and then separating and purifying to obtain an intermediate A-6; A structural formula of the intermediate A-6 is as follows: S4, dissolving the compound A-6 in ethyl acetate, adding stannous chloride dihydrate and concentrated hydrochloric acid, and heating and reacting at 65-75 °C for 10-24 h, and then separating and purifying to obtain the fluorescent probe.

[0033] A synthesis route of the fluorescent probe is as follows: .

[0034] ​​​The mechanism of the fluorescence-enhanced probe for identifying methylglyoxal in the present application is as follows: taking tricyanofuran as a fluorescent group and o-phenylenediamine as a recognition group, the probe is in a fluorescence quenching state, under the action of methylglyoxal, a 2-methylquinoxaline structure is generated, the transformation of the structure can inhibit the PET process in the probe molecule, resulting in the opening of fluorescence, thereby achieving the detection effect. By monitoring the fluorescence signal at 530 nm and the fluorescence intensity of the product, the concentration of methylglyoxal can be detected in real time.

[0035] In addition, the present specific embodiment also proposes an application of the above-mentioned fluorescence probe or the fluorescence probe prepared by the above-mentioned preparation method in detecting methylglyoxal in an environment or a biological sample, and the application includes imaging detection of cells, zebrafish or Arabidopsis thaliana, and the cells are suitable for non-therapeutic purposes.

[0036] As shown in Table 1, compared with the reported fluorescence probes for methylglyoxal, the fluorescence probe involved in the present application has the following advantages: the fluorescence probe of the present application has a larger Stokes shift, stronger anti-interference ability and strong sample penetration; the fluorescence probe of the present application has a lower detection limit and high sensitivity, and can be used for detection of lower concentration of MGO; the fluorescence probe of the present application can be used for detection of endogenous methylglyoxal in living cells and zebrafish, and can realize Arabidopsis thaliana MGO in vivo fluorescence imaging, and observe the distribution imaging and detection of MGO in vivo. It has application prospect related to methylglyoxal.

[0037] Table 1 Through comprehensive analysis of the above problems, the enhanced fluorescence probe for detecting methylglyoxal can solve the following problems: 1) improve the sensitivity of the probe and reduce its detection limit; 2) make the probe have a larger Stokes shift and improve its anti-interference ability; 3) improve its application value, and the probe can be applied to detection and imaging of endogenous methylglyoxal in living cells and in vivo of animals and plants, and realize in situ fluorescence imaging of plants in vivo.

[0038] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application is further described in detail below in combination with the drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application, and are not used to limit the present application.

[0039] In the present application, "some embodiments", "the present embodiment" and examples and the like are described, which describe a subset of all possible embodiments, but it can be understood that "some embodiments" can be the same subset or different subset of all possible embodiments, and can be combined with each other without conflict.

[0040] If the application file contains similar descriptions of "first / second", the following description is added. In the following description, the terms "first / second / third" are only used to distinguish similar objects, and do not represent a specific order of the objects. Understandably, "first / second / third" can be interchanged in a specific order or sequence as allowed, so that the embodiments described herein can be implemented in an order other than that illustrated or described herein.

[0041] In this embodiment, the term "and / or" is only used to describe the association relationship of the associated objects, which means that there can be three relationships, for example, object A and / or object B, which can represent three cases: object A exists alone, object A and object B exist together, and object B exists alone.

[0042] Hereinafter, the embodiments of the present application will be described. The embodiments described below are exemplary and are used only to explain the present application, and cannot be understood as a limitation of the present application. If the specific technology or condition is not specified in the embodiments, it is carried out according to the technology or condition described in the literature in the art or according to the product manual. If the reagent or instrument used is not specified by the manufacturer, it is a conventional product that can be obtained by purchase.

[0043] Example 1 This embodiment proposes a preparation method of a fluorescent probe for detecting methylglyoxal, and the specific steps are as follows: Synthesis of intermediate A-2: The concentrated nitric acid (0.5 mL) was slowly added to the concentrated sulfuric acid (2.5 mL) with temperature control at 0-5 °C, and the mixture was uniformly mixed to obtain a mixed solution. Then, 4-acetylamino benzaldehyde (1 g, 6.13 mmol) was slowly added to the reaction bottle containing the above-mentioned mixed solution, and the solid was gradually dissolved. After the reaction was completed, the reaction was transferred to an ice-water mixture, and the solid was precipitated to obtain a yellow crude product, which was then purified by ethanol recrystallization to obtain 1.1 g of yellow solid with a yield of 80.8 %. 1 HNMR (400 MHz, Chloroform-d) δ 10.62 (s, 1H), 9.98 (s, 1H), 9.03 (d, J = 8.8Hz, 1H), 8.73 (s, 1H), 8.15 (d, J = 10.8 Hz, 1H), 2.35 (s, 3H). Synthesis of Intermediate A-4: 3-Hydroxy-3-methylbutan-2-one (500 mg, 4.9 mmol), malononitrile (647 mg, 9.8 mmol) and potassium carbonate (11 mg, 0.015 mmol) were heated and stirred in an oil bath at 80 °C for 20 min. Then 15 mL of ethanol was added to the mixture and stirred for 20 min. The precipitate was filtered, washed with ethanol solution and dried under vacuum to obtain 402 mg of yellowish solid with a yield of 41 %. 1 H NMR (400 MHz, Chloroform-d) δ 2.37 (s, 3H), 1.63 (s, 6H).

[0044] Synthesis of Intermediate A-5: Intermediate A-2 (205 mg, 1.2 mmol) and Intermediate A-4 (199 mg, 1 mmol) were dissolved in 10 mL of ethanol. To the mixture, 50 μL of piperidine was added and refluxed at 80 °C for 1 h. After the reaction was completed, the solvent was removed by distillation under reduced pressure to obtain an orange solid. The solid was washed with ethanol and dried under vacuum to obtain 440 mg of orange-red solid with a yield of 66 %. 1 H NMR (400 MHz, DMSO-d6) δ 10.52 (s, 1H), 8.55 (s, 1H), 8.26 (d, J = 8.6 Hz, 1H), 7.93 (d, J = 16.6 Hz, 1H), 7.81 (d, J = 8.6 Hz, 1H), 7.31 (d, J = 16.6 Hz, 1H), 2.12 (s, 3H), 1.82 (s, 6H). Synthesis of Intermediate A-6: Intermediate A-5 (389 mg, 1 mM) was dissolved in 10 mL of ethyl acetate. Then 1 mL of concentrated hydrochloric acid was added to the solution and refluxed at 80 °C for 2 h. After the reaction was completed, it was neutralized with sodium hydroxide solution. Subsequently, a large amount of solid was precipitated in the solution. The precipitate was filtered, washed with dichloromethane and dried under vacuum to obtain 274 mg of dark red solid with a yield of 78 %. 1 H NMR (400 MHz, DMSO-d6) δ 8.61 (s, 1H), 8.23 (s, 2H), 8.05 (d, J = 9.0 Hz, 1H), 7.92 (d, J = 16.4 Hz, 1H), 7.13 (d, J = 8.9 Hz, 1H), 7.02 (d, J = 16.3 Hz, 1H), 1.80 (s, 6H).

[0045] Synthesis of fluorescent probe TCF-OPD: Intermediate A-6 (347 mg, 1 mM) was dissolved in 10 mL tetrahydrofuran (THF), then stannous chloride dihydrate (2256 mg, 10 mM) and 1 mL concentrated hydrochloric acid were added, heated to 70 °C, and refluxed for 12 h. After the reaction was completed, the reaction solution was cooled to room temperature, and THF was removed by reduced pressure distillation. Then 1 mL saturated brine was added, the pH value was adjusted to 8-10 with 15 % sodium hydroxide solution, the black crude product was extracted with ethyl acetate, and the black product was obtained by reduced pressure distillation. The crude product was purified by column chromatography (eluent: petroleum ether / ethyl acetate = 10 / 1 to 1 / 2, volume ratio) to obtain 269 mg of black solid, yield 77 %, 1 H NMR (400 MHz, DMSO-d6) δ 7.76 (d, J = 15.5Hz, 1H), 7.16 (d, J = 8.4 Hz, 1H), 7.06 (s, 1H), 6.69 (d, J = 15.5 Hz, 1H),6.61 (d, J = 8.3 Hz, 1H), 6.53 (s, 2H), 5.00 (s, 2H), 1.74 (s, 6H).

[0046] The HRMS chart of the fluorescent probe obtained in this example is shown in 13 C NMR as Figure 2 shown. 13 C NMR (101 MHz, DMSO-d6) δ 177.70, 175.34, 150.47, 145.43, 135.51, 128.09, 124.19, 114.24, 114.06, 113.45, 112.81, 112.64, 107.63, 98.15, 90.88, 50.33, 26.34.

[0047] The HRMS chart of the fluorescent probe obtained in this example is shown in Figure 3 HRMS (ESI) calcd for C 18 H 15 N5O[M+H+] 318.1349, found 318.1346. Example 2 Test the response effect of the fluorescent probe obtained in Example 1 on methylglyoxal: A stock solution of 10 mM of the compound TCF-OPD in Example 1 was prepared using DMSO; a stock solution of 100 mM of methylglyoxal was prepared using ultrapure water. 1 μL of the stock solution of the compound TCF-OPD and 999 μL of PBS (pH = 7.4) buffer were placed in a cuvette, and the fluorescence spectrum at 410 nm was detected; 1 μL of the stock solution of the compound TCF-OPD, 998 μL of PBS (pH = 7.4) buffer and 1 μL of the methylglyoxal solution were placed in a 37 °C water bath for incubation, and then transferred to a cuvette, and the fluorescence spectrum at 410 nm was detected. The results are shown in Figure 1. Figure 4 As can be seen from Figure 1, the probe has no fluorescence, and the fluorescence intensity of the probe at 530 nm gradually increases with increasing reaction time. After the reaction reaches 45 min, the fluorescence intensity reaches a plateau, indicating that the probe is a fluorescence-enhanced probe for recognizing methylglyoxal. When the reaction time reaches 50 min, the fluorescence intensity no longer increases, and the reaction is complete.

[0048] Example 3 The fluorescence spectrum of the fluorescence probe obtained in Example 1 in reaction with methylglyoxal of different concentrations was tested: The above-mentioned solution of the compound TCF-OPD (working concentration 10 μM), PBS (pH = 7.4) buffer and methylglyoxal of different working concentrations (0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 160, 180, 200 μM) were added to a cuvette, and the fluorescence spectrum of the fluorescence at 530 nm after reaction for 45 min was detected, as shown in Figure 2. Figure 5 As can be seen from Figure 2, the fluorescence intensity of the reaction system at 530 nm increases with increasing concentration of methylglyoxal, indicating that the probe can detect methylglyoxal of different concentrations.

[0049] Example 4 The detection limit of the fluorescence probe obtained in Example 1 for detecting methylglyoxal was determined: The above-mentioned solution of the compound TCF-OPD (working concentration 10 μM), PBS (pH = 7.4) buffer and methylglyoxal of different working concentrations (0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120 μM) were added to a cuvette, and the fluorescence intensity at 530 nm after reaction for 45 min and the linear relationship with methylglyoxal were determined, as shown in Figure 3. Figure 6As shown, the fluorescence intensity at 530 nm and methylglyoxal showed a good linear relationship, indicating that the fluorescence probe could quantitatively detect methylglyoxal in the range of (0-120 μM). At the same time, according to the linear relationship graph, the detection limit of the probe for methylglyoxal was calculated to be 0.14 μM, indicating that the probe was relatively sensitive and could detect lower concentrations of methylglyoxal.

[0050] Example 5 Test the selectivity of the fluorescence probe obtained in Example 1 to recognize methylglyoxal: Into the cuvette, respectively add 1 μL of the fluorescence probe obtained in Example 1 (10 μM, dissolved in 37 ℃ pH 7.4 PBS buffer solution) and different analyte mixtures (a: blank; b: Cys (100 μM); c: Hcy (100 μM); d: GSH (100 μM); e: Glu (100 μM); f: Gly (100 μM); g: S 2- (100 μM); h: HCO3 - (100 μM); i: NO3 - (100 μM); j: NO2 - (100 μM); k: SO4 2- (100 μM); l: Ba 2+ (100 μM); m: Ca 2+ (100 μM); n: Fe 3+ (100 μM); o: Zn 2+ (100 μM); p: Mg 2+ (100 μM); q: OA (100 μM); r: AA (100 μM); s: NO (100 μM); t: HCHO (100 μM); u: MGO (100 μM), test the fluorescence intensity at 530 nm after 45 min of each mixture. As Figure 7 As shown, in the presence of various anions, metal cations and amino acids, the fluorescence signal of the fluorescence probe was basically unchanged, and after the addition of methylglyoxal, its fluorescence was significantly enhanced, indicating that the fluorescence probe had high selectivity for methylglyoxal.

[0051] Example 6 The fluorescent probe obtained in Example 1 (10 μM) was co-incubated with HeLa cells, and after washing with PBS, the cells were imaged under a microscope. The first group was a control group, which was incubated with the fluorescent probe for 30 min. The second group was a curcumin group, which was incubated with different concentrations (50, 100, 200 μM) of curcumin solution, and then incubated with the fluorescent probe. The third group was a curcumin + N-acetylcysteine group. N-acetylcysteine (NAC) is a common MGO scavenger that can remove MGO in cells. The cells were incubated with 200 μM curcumin, then incubated with 200 μM N-acetylcysteine, and finally incubated with the fluorescent probe. The results are shown in FIG. 2. The fluorescence intensity after incubation with curcumin was higher than that of the control group, and the fluorescence increased with the increase of the concentration of curcumin. After incubation with N-acetylcysteine, the fluorescence intensity decreased. This indicates that the probe can detect changes in the content of MGO in cells regulated by curcumin and N-acetylcysteine. Figure 8

[0052] Example 7 The fluorescent probe obtained in Example 1 was used to detect methyglyoxal in live zebrafish.

[0053] Zebrafish larvae grown for 0-7 days were cultured in a 6-well plate and divided into three groups. The first group was a control group, which was incubated with the fluorescent probe for 30 min. The second group was a curcumin group, which was incubated with different concentrations (50, 100, 200 μM) of curcumin solution, and then incubated with the fluorescent probe. The third group was a curcumin + N-acetylcysteine group. The cells were incubated with 200 μM curcumin, then incubated with 200 μM N-acetylcysteine, and finally incubated with the fluorescent probe. As shown in FIG. 8, the fluorescence intensity of zebrafish increased with the increase of the concentration of curcumin, and after the addition of the scavenger for pretreatment, the zebrafish had almost no fluorescence, indicating that the probe can specifically detect endogenous methyglyoxal activity in a living organism. Figure 9

[0054] Example 8 The fluorescent probe obtained in Example 1 was used to detect methyglyoxal in Arabidopsis thaliana root tip tissue.

[0055] Arabidopsis grown for 9 days was cultured and divided into three groups. The first group was a control group, and the root tip was incubated with the fluorescent probe for 30 min. The second group was a methyglyoxal group, and the root tip was incubated with 100 μM methyglyoxal solution, and then incubated with the fluorescent probe. The third group was a methyglyoxal + N-acetylcysteine group. The root tip was incubated with 100 μM methyglyoxal, then incubated with 200 μM N-acetylcysteine, and finally incubated with the fluorescent probe. As shown in FIG. 12, the fluorescence intensity of the root tip increased with the increase of the concentration of methyglyoxal, and after the addition of the scavenger for pretreatment, the root tip had almost no fluorescence, indicating that the probe can specifically detect endogenous methyglyoxal activity in a living organism. Figure 10 ​​As shown, the fluorescence intensity of Arabidopsis root tip tissue increased with the addition of methylglyoxal, while the fluorescence of Arabidopsis decreased after pretreatment with a scavenger, indicating that the probe can quantitatively analyze the content of methylglyoxal in plants.

[0056] Example 9 Distribution imaging and detection of in situ live MGO in Arabidopsis thaliana obtained from Example 1.

[0057] Nine-day-old Arabidopsis thaliana plants were divided into three groups. The first group was the control group, where plants were incubated with a fluorescent probe for 30 minutes. The second group was the methylglyoxal group, where plants were incubated with 100 μM methylglyoxal solution, followed by incubation with a fluorescent probe. The third group was the N-acetylcysteine ​​methylglyoxal+ group, where plants were incubated with 100 μM methylglyoxal, then with 200 μM N-acetylcysteine ​​solution, and finally with a fluorescent probe. Figure 11 As shown, the fluorescence intensity of Arabidopsis thaliana increased with the addition of methylglyoxal, while the fluorescence of Arabidopsis thaliana decreased after pretreatment with a scavenger. This indicates that the probe can quantitatively analyze the content of methylglyoxal in plants and monitor the distribution of MGO in plants.

[0058] The fluorescent probe of this invention possesses advantages such as low fluorescence background, good chemical stability, high selectivity, large Stokes shift, and high sensitivity. The probe itself is in a fluorescence-quenched state, and upon interaction with methylglyoxal (MGO), it exhibits strong fluorescence, with an enhancement of approximately 23 times. This fluorescent probe can be used for linear detection of MGO in the concentration range of 0-120 μM, with a detection limit of 0.14 μM, exhibiting high sensitivity and enabling qualitative and quantitative detection of MGO. This probe can detect MGO in cells and zebrafish, and can also non-destructively image and detect the distribution of MGO in living Arabidopsis thaliana. It shows promising applications in in-situ imaging of MGO in plants and in studying the accumulation of MGO in various parts of living plants and its regulatory mechanisms on plant growth, development, and stress responses.

[0059] The specific embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any other corresponding changes and modifications made in accordance with the technical concept of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A fluorescent probe for recognizing methylglyoxal, characterized by, The structural formula is: (Ⅰ)。 2. The fluorescent probe according to claim 1, wherein The product after the fluorescent probe responds to methylglyoxal has a maximum emission wavelength at 530 nm, and has green fluorescence.

3. The fluorescent probe according to claim 2, wherein The structural formula of the product is: (Ⅱ)。 4. A method for preparing the fluorescent probe according to any one of claims 1 to 3, characterized in that, The method comprises the following steps: S1, mixing concentrated nitric acid and concentrated sulfuric acid to obtain a mixed solution, then mixing and reacting p-acetamidobenzaldehyde and the mixed solution, and then separating and purifying to obtain intermediate A-2; dissolving 3-hydroxy-3-methyl-2-butanone in anhydrous ethanol, adding malononitrile and potassium carbonate, and heating to react, and then separating and purifying to obtain intermediate A-4; The structural formula of the intermediate A-2 is: ; The structural formula of the intermediate A-4 is: ; S2, dissolving the intermediate A-2 and the intermediate A-4 in anhydrous acetonitrile, adding piperidine, and refluxing to react, and then separating and purifying to obtain intermediate A-5; The structural formula of the intermediate A-5 is: ; S3, dissolving the intermediate A-5 in ethyl acetate, adding concentrated hydrochloric acid, and refluxing to react, and then separating and purifying to obtain intermediate A-6; The structural formula of the intermediate A-6 is: ; S4, dissolving the compound A-6 in ethyl acetate, adding stannous chloride dihydrate and concentrated hydrochloric acid, and heating to react, and then separating and purifying to obtain the fluorescent probe.

5. The method for preparing the fluorescent probe according to claim 4, characterized in that, In step S1, the mixing and reaction time is 1-1.5h; and / or, in step S2, the refluxing reaction time is 1-3h; and / or, in step S3, the refluxing reaction time is 22-25h; and / or, in step S4, the heating reaction time is 10-24h.

6. The method for preparing the fluorescent probe according to claim 4, characterized in that, In step S1, the heating reaction temperature is 75-85 °C.

7. The method for preparing the fluorescent probe according to claim 4, characterized in that, In step S2 or step S3, the refluxing reaction temperature is 75-85 °C.

8. The method for preparing the fluorescent probe according to claim 4, characterized in that, In step S4, the heating reaction temperature is 65-75 °C.

9. Use of the fluorescent probe of any one of claims 1-3 or the fluorescent probe prepared by the preparation method of any one of claims 4-8 in detecting methylglyoxal in an environment or a biological sample.

10. Use according to claim 9, characterized in that, The use comprises imaging detection of cells, zebrafish or Arabidopsis thaliana, and the cells are suitable for non-therapeutic purposes.

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