A fluorescent probe for detecting methylglyoxal with a large Stokes shift, and its preparation method and application

Abstract

The present invention discloses a fluorescent probe for detecting methylglyoxal with a large Stokes shift, a preparation method thereof, and an application thereof, and belongs to the field of chemical analysis and detection technology. The fluorescent probe for detecting methylglyoxal has the structural formula: . In addition, the present invention also proposes a method for preparing the above-mentioned fluorescent probe for detecting methylglyoxal, comprising: dissolving compound C-3 in a first solvent, then adding concentrated hydrochloric acid and heating to 75-85°C for reflux reaction to obtain an intermediate BH-PDO; dissolving the intermediate BH-PDO in a second solvent, then adding stannous chloride dihydrate and concentrated hydrochloric acid for reflux reaction to obtain the fluorescent probe. In addition, the present invention also proposes a fluorescent probe prepared by the above-mentioned preparation method. In addition, the present invention also proposes the application of a fluorescent probe in detecting methylglyoxal. The fluorescent probe proposed by the present invention has a large Stokes shift and strong anti-interference ability, and can specifically detect methylglyoxal.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical analysis and detection, and in particular to a fluorescent probe for detecting methylglyoxal with a large Stokes shift, and a preparation method and application thereof. Background Art

[0002] Methylglyoxal (MGO) is a reactive α,β-dicarbonyl ketoaldehyde primarily produced in physiological systems through metabolic pathways such as glucose, alcohol, and protein. MGO, a product of the spontaneous conversion of trisonate phosphate, is an endogenous dicarbonyl metabolite that plays a key role in plant growth and development, signal transduction, and non-stress responses. However, MGO is a double-edged sword. High concentrations of MGO can produce significant toxic effects on plant cells, such as inhibiting seed germination, hindering normal root development, and interfering with photosynthesis. Clearly, the concentration of MGO in plants plays a critical role in ensuring the optimal functioning of plant physiological processes. Furthermore, MGO concentration is closely correlated with the ability of plants to withstand abiotic stresses such as drought, salinity, and extreme temperatures. Therefore, MGO is considered a potential biochemical marker for assessing plant stress resilience. In summary, the detection of MGO in plants is of great significance for further understanding the mechanisms of plant growth and development and stress responses.

[0003] Currently, methods for detecting methylglyoxal include high-performance liquid chromatography, gas chromatography, electrochemical methods, ultraviolet spectrophotometry, and fluorescent probe analysis. Compared to traditional analytical methods, fluorescence imaging technology can provide more intuitive and accurate data support, making it a powerful tool for studying the distribution of methylglyoxal in organisms and its interactions with other metabolites. To date, several fluorescent probes have been developed to detect methylglyoxal, but almost all of these probes have been developed for animal and cell studies, with few probes being used to study the impact of methylglyoxal content on plant growth processes. Therefore, there is an urgent need to develop a new fluorescent probe that can detect methylglyoxal content in animals and plants.

[0004] Based on a comprehensive analysis of the above issues, the following problems need to be solved for the enhanced fluorescent probe for detecting methylglyoxal: 1) improving the sensitivity of the probe and lowering its detection limit; 2) making the probe have a larger Stokes shift to improve its anti-interference ability; 3) enabling the probe to be loaded on a test strip for convenient detection; and 4) improving its application value. The probe can be applied to the detection and imaging of endogenous methylglyoxal in living cells and living plants and animals. Summary of the Invention

[0005] The purpose of the present invention is to overcome the above technical deficiencies, provide a fluorescent probe for detecting methylglyoxal with a large Stokes shift, and a preparation method and application thereof, so as to solve the technical problem of how to increase the Stokes shift of fluorescent detection of methylglyoxal in the prior art.

[0006] In order to achieve the above technical objectives, the technical solution of the present invention provides a fluorescent probe for detecting methylglyoxal, the structural formula of which is shown in formula (I):

[0007] .

[0008] In addition, the present invention also provides a method for preparing the fluorescent probe for detecting methylglyoxal, comprising the following steps:

[0009] S1. Dissolve compound C-3 in a first solvent, then add concentrated hydrochloric acid and heat to 75-85° C. for reflux reaction to obtain the intermediate BH-PDO;

[0010] S2, dissolving the intermediate BH-PDO in a second solvent, then adding stannous chloride dihydrate and concentrated hydrochloric acid, heating to 75-85° C. for reflux reaction to obtain the fluorescent probe;

[0011] The structural formula of the compound C-3 is as follows:

[0012] ;

[0013] The structural formula of the intermediate BH-PDO is as follows:

[0014] .

[0015] In any embodiment, in step S1, the first solvent is ethyl acetate; and / or, in step S2, the second solvent is ethyl acetate; and / or, in step S1, the reflux reaction is performed for 4-8 hours; and / or, in step S2, the reflux reaction is performed for 4-8 hours; and / or, in step S1, the compound C-3 is prepared by the following steps:

[0016] Intermediate C-2 and intermediate D-2 are dissolved in a third solvent, followed by addition of piperidine, and the mixture is refluxed at 75-85°C to obtain intermediate C-3;

[0017] The structural formula of the intermediate C-2 is: The structural formula of the compound D-2 is: ; and / or, the intermediate C-2 is prepared by the following steps: dissolving compound C-1 in a fourth solvent and adding 1-iodopropane, and reacting under reflux at 75-85° C. to obtain the intermediate C-2;

[0018] The structural formula of the compound C-1 is: ; and / or, the intermediate D-2 is prepared by the following steps:

[0019] Mix concentrated nitric acid and concentrated sulfuric acid to obtain a mixed solution, and then slowly add the mixed solution dropwise to the reaction vessel containing compound D-1 to react and obtain the intermediate D-2;

[0020] The structural formula of the compound D-1 is: .

[0021] In any embodiment, the third solvent is anhydrous acetonitrile; and / or the fourth solvent is anhydrous acetonitrile.

[0022] In addition, the present invention also provides a fluorescent probe prepared by the above preparation method.

[0023] In addition, the present invention also provides the fluorescent probe for detecting methylglyoxal or the use of the fluorescent probe in detecting methylglyoxal in the environment or biological samples.

[0024] In any embodiment, the product produced by the reaction of the fluorescent probe (I) with methylglyoxal has a maximum emission wavelength at 625 nm and exhibits red fluorescence.

[0025] In any embodiment, the structural formula of the product is as shown in formula (II):

[0026] .

[0027] In any embodiment, the method comprises qualitative and quantitative detection of methylglyoxal in aqueous solution; or imaging detection of cells, zebrafish or Arabidopsis, wherein the cells are suitable for non-therapeutic purposes.

[0028] In addition, the present invention also provides a fluorescent reagent detection kit, which includes the above-mentioned fluorescent probe or the fluorescent probe prepared by the above-mentioned preparation method.

[0029] Compared with the prior art, the present invention has the following beneficial effects: the fluorescent probe for detecting methylglyoxal proposed by the present invention has a large Stokes shift (255 nm), strong anti-interference ability, and can specifically detect methylglyoxal. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The fluorescent probe obtained in Example 1 of the present invention 1 H NMR spectrum.

[0031] Figure 2 The fluorescent probe obtained in Example 1 of the present invention 13 C NMR.

[0032] Figure 3 This is the HRMS spectrum of the fluorescent probe obtained in Example 1 of the present invention.

[0033] Figure 4 This is a fluorescence spectrum diagram of the fluorescent probe detecting methylglyoxal in response to Example 2 of the present invention.

[0034] Figure 5 The fluorescence spectra of the fluorescent probe of Example 3 of the present invention reacting with methylglyoxal at different concentrations are shown.

[0035] Figure 6 This is a test diagram of the detection limit of the fluorescent probe in Example 4 of the present invention for detecting methylglyoxal in a low concentration range.

[0036] Figure 7 This is a histogram of the fluorescence response of the fluorescent probe of Example 5 of the present invention reacting with different analytes.

[0037] Figure 8 The fluorescent probe of Example 6 of the present invention is fixed on a test strip to conveniently detect methylglyoxal.

[0038] Figure 9 This is a fluorescence imaging diagram of the fluorescent probe of Example 7 of the present invention detecting methylglyoxal in living cells.

[0039] Figure 10 This is a fluorescence imaging diagram of the fluorescent probe in Example 8 of the present invention detecting methylglyoxal in living zebrafish.

[0040] Figure 11 This is a fluorescence imaging image of methylglyoxal in the root tip tissue of Arabidopsis thaliana detected by the fluorescent probe in Example 9 of the present invention. DETAILED DESCRIPTION

[0041] The "range" disclosed in this application is defined in the form of a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundaries of a particular range. The range defined in this way can be inclusive or exclusive of the end values, and can be arbitrarily combined, i.e., any lower limit can be combined with any upper limit to form a range. For example, if a range of 60 to 120 and 80 to 110 is listed for a particular parameter, it is understood that a range of 60 to 110 and 80 to 120 is also expected. In addition, if the minimum range values ​​listed are 1 and 2, and if the maximum range values ​​listed are 3, 4, and 5, then the following ranges can all be expected: 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4, and 2 to 5. In this application, unless otherwise specified, the numerical range "a to b" represents an abbreviation of any real number combination between a and b, wherein a and b are both real numbers. For example, a numerical range of "0-5" indicates that all real numbers between "0-5" are listed herein, and "0-5" is simply an abbreviation for these numerical combinations. Furthermore, stating that a parameter is an integer ≥ 2 is equivalent to disclosing that the parameter is, for example, an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0042] Unless otherwise specified, the terms "include" and "comprising" used in this application may be open-ended or closed-ended. For example, "include" and "comprising" may mean that other components not listed may also be included or that only the listed components are included.

[0043] Unless otherwise specified, the term "or" is used in this application to be inclusive. For example, the phrase "A or B" means "A, B, or both A and B." More specifically, the condition "A or B" is satisfied if any of the following conditions are met: 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 both A and B are true (or exist).

[0044] As shown in the table below, compared with previously reported fluorescent probes for methylglyoxal, the fluorescent probe of the present invention has the following advantages: the fluorescent probe of the present invention has a larger Stokes shift, stronger anti-interference ability, and strong sample penetration; the fluorescent probe of the present invention has a lower detection limit and high sensitivity, and can be used to detect relatively low concentrations of MGO; the fluorescent probe of the present invention can be loaded on a test strip for convenient detection; the fluorescent probe of the present invention can be used for the detection and imaging of endogenous methylglyoxal in living cells, living zebrafish, and Arabidopsis thaliana, and has application prospects in detecting methylglyoxal-related substances.

[0045]

[0046] This embodiment provides a fluorescent probe for detecting methylglyoxal with a large Stokes shift, and its molecular formula is C 25 H 28 IN3, whose structural formula is shown in formula (I):

[0047] .

[0048] This specific embodiment also provides a method for preparing the above-mentioned fluorescent probe for detecting methylglyoxal with a large Stokes shift, comprising the following steps:

[0049] S1. Dissolve compound C-3 in a first solvent, then add concentrated hydrochloric acid and heat to 75-85° C. for reflux reaction to obtain the intermediate BH-PDO;

[0050] S2, dissolving the intermediate BH-PDO in a second solvent, then adding stannous chloride dihydrate and concentrated hydrochloric acid, heating to 75-85° C. for reflux reaction to obtain the fluorescent probe;

[0051] The structural formula of the compound C-3 is as follows:

[0052] ;

[0053] The structural formula of the intermediate BH-PDO is as follows:

[0054] .

[0055] In some embodiments, in step S1, the first solvent is ethyl acetate; in step S2, the second solvent is ethyl acetate; in step S1, the reflux reaction is performed for 4-8 hours; in step S2, the reflux reaction is performed for 4-8 hours.

[0056] In some embodiments, in step S1, the compound C-3 is prepared by the following steps:

[0057] Intermediate C-2 and intermediate D-2 are dissolved in a third solvent, followed by addition of piperidine, and the mixture is refluxed at 75-85°C to obtain intermediate C-3;

[0058] The structural formula of the intermediate C-2 is: The structural formula of the compound D-2 is: .

[0059] In some embodiments, the intermediate C-2 is prepared by the following steps:

[0060] Dissolve compound C-1 in a fourth solvent and add 1-iodopropane, and reflux at 75-85° C. to obtain the intermediate C-2;

[0061] The structural formula of the compound C-1 is: ;

[0062] The intermediate D-2 is prepared by the following steps:

[0063] Mix concentrated nitric acid and concentrated sulfuric acid to obtain a mixed solution, and then slowly add the mixed solution dropwise to the reaction vessel containing compound D-1 to react and obtain the intermediate D-2;

[0064] The structural formula of the compound D-1 is: .

[0065] In some embodiments, the third solvent is anhydrous acetonitrile; and the fourth solvent is anhydrous acetonitrile.

[0066] The synthetic route of the fluorescent probe is as follows:

[0067]

[0068] In addition, this specific embodiment also proposes a fluorescent probe, which is prepared by the above preparation method.

[0069] In addition, this specific embodiment also proposes the above-mentioned fluorescent probe for detecting methylglyoxal or the use of the above-mentioned fluorescent probe in detecting methylglyoxal in the environment or biological samples.

[0070] In some embodiments, the product of the reaction between the fluorescent probe (I) and methylglyoxal has a maximum emission wavelength at 625 nm and exhibits red fluorescence.

[0071] In some embodiments, the product has the formula C 28 H 28 IN3, the structural formula is shown in formula (II):

[0072] .

[0073] In some embodiments, the method includes qualitative and quantitative detection of methylglyoxal in aqueous solution; or imaging detection of cells, zebrafish or Arabidopsis, wherein the cells are suitable for non-therapeutic purposes.

[0074] The reaction formula of fluorescent probe (I) and methylglyoxal is as follows:

[0075]

[0076] In addition, this specific embodiment also provides a fluorescent reagent detection kit, which includes the above-mentioned fluorescent probe or the fluorescent probe prepared by the above-mentioned preparation method.

[0077] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0078] In the present invention, references to “some embodiments”, “this embodiment”, examples, etc. describe a subset of all possible embodiments, but it can be understood that “some embodiments” may be the same subset or different subsets of all possible embodiments, and may be combined with each other without conflict.

[0079] If similar descriptions of "first / second" appear in the application documents, the following explanation is added. In the following description, the terms "first\second\third" are merely used to distinguish similar objects and do not represent a specific order for the objects. It is understandable that "first\second\third" can be interchanged with the specific order or sequence where permitted, so that the embodiments described herein can be implemented in an order other than that illustrated or described herein.

[0080] In this embodiment, the term "and / or" is merely a description of the association relationship between associated objects, indicating that three relationships may exist. For example, object A and / or object B may represent three situations: object A exists alone, object A and object B exist at the same time, and object B exists alone.

[0081] Below, the embodiment of the present application is described. The embodiment described below is exemplary and is only used to explain the present application, and is not to be construed as limiting the present application. Where specific techniques or conditions are not specified in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. Reagents or instruments used that do not specify the manufacturer are conventional products that can be obtained commercially.

[0082] Example 1

[0083] This embodiment provides a method for preparing a fluorescence-enhanced probe for detecting methylglyoxal, and the specific steps are as follows:

[0084] Synthesis of Intermediate C-2: Compound C-1 (1054.2 mg, 5 mmol) was dissolved in 15 mL of anhydrous acetonitrile. 1-Iodopropane (3.5 mL, 35 mmol) was slowly added and refluxed at 80°C for 17 h. After completion of the reaction, the mixture was cooled to room temperature and the solvent was removed by distillation under reduced pressure to yield a dark green solid. This solid was then washed with 1 mL of methanol and 5 mL of anhydrous ether to remove impurities. The resulting solid was then dissolved in 2 mL of dichloromethane, and 10 mL of petroleum ether was added. The solvent was then removed by distillation under reduced pressure to yield 1590 mg of a light green solid, Intermediate C-2, in an 83% yield. 1H NMR (400 MHz, DMSO-d6) δ 8.40 (d, J = 8.3 Hz,1H), 8.32 (d, J = 8.9 Hz, 1H), 8.23 ​​(t, J = 9.4 Hz, 2H), 7.83-7.72 (m, 2H),4.61 (t, J = 7.4 Hz, 2H), 3.00 (s, 3H), 1.96 (q, J = 7.4 Hz, 2H), 1.79 (s, 6H), 1.05 (t, J = 7.3 Hz, 3H).

[0085] Synthesis of intermediate D-2: Concentrated nitric acid (0.375 mL) was slowly added dropwise to concentrated sulfuric acid (2.25 mL) at a temperature of 0-5°C and mixed thoroughly. The mixed solution was then slowly added dropwise to a reaction flask containing 4-acetamidobenzaldehyde (1 g, 6.13 mmol), and the solid gradually dissolved. After the reaction was complete, the reactants were transferred to an ice-water mixture to precipitate a solid to obtain a crude yellow product. The crude product was then purified by column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 5 to 1 / 12, v / v) to obtain 1.1 g of intermediate D-2 as a yellow solid with a yield of 80.8%. 1 H NMR (400 MHz, DMSO-d6) δ 10.61 (s,1H), 9.98 (s, 1H), 8.44 (s, 1H), 8.15 (d, J=8.0 Hz, 1H), 7.87 (d. J=8.0 Hz,1H), 2.14 (s, 3H).

[0086] Synthesis of intermediate C-3: Intermediate C-2 (380 mg, 1 mmol) and intermediate D-2 (312 mg, 1.5 mmol) were dissolved in 10 mL of anhydrous acetonitrile, and 80 μL of piperidine was added thereto. The mixture was stirred at room temperature for 1 min until the solution turned yellow. The mixture was further stirred at 80°C for 1 h. After the reaction, the solvent was removed by distillation under reduced pressure to obtain an orange-yellow solid powder. The crude product was then purified by silica gel column chromatography (eluent: from dichloromethane / methanol = 200 / 1 to 20 / 1, v / v) to obtain 0.454 g of intermediate C-3 as a yellow solid with a yield of 81%. 1H NMR (400 MHz, DMSO-d6) δ 10.64 (s, 1H), 8.87 (s, 1H), 8.68-8.58 (m, 2H), 8.49 (s, 1H), 8.32 (d, J=9.0 Hz, 1H), 8.27-8.16 (m, 2H),7.90 (d, J=8.7 Hz, 1H), 7.86-7.73 (m, 3H), 4.86 (t, J=7.4 Hz, 2H), 2.16 (s,3H), 2.05 (s, 6H), 1.97 (q, J=7.2 Hz, 2H), 1.04 (t, J=7.3 Hz, 3H).

[0087] Synthesis of intermediate BH-PDO: Compound C-3 (380 mg, 0.67 mmol) was weighed and dissolved in 10 mL of ethyl acetate. After stirring until the solid was completely dissolved, 1 mL of concentrated hydrochloric acid was added and heated to 80°C for reflux reaction for 4 h. After the reaction, the reaction solution was cooled to room temperature and the ethyl acetate was removed by rotary evaporation. 1 mL of saturated brine was added and the pH was adjusted to 8-10 with 15% sodium hydroxide solution. The product was extracted with ethyl acetate and distilled under reduced pressure to obtain a red crude product. The crude product was purified by column chromatography (eluent: dichloromethane / methanol = 100 / 1 to 10 / 1, v / v) to obtain 269 mg of the intermediate BH-PDO as a red solid with a yield of 77%. 1 H NMR (400 MHz, DMSO-d6) δ 8.97 (d, J=2.0 Hz, 1H), 8.62 (d, J=16.0 Hz,1H), 8.55-8.31 (m, 4H), 8.28 (d, J=8.9 Hz, 1H), 8.21 (d, J=8.2 Hz, 1H), 8.11(d, J=9.0 Hz, 1H), 7.81 (ddd, J=8.3, 6.8 Hz, 1H), 7.72 (t, J=7.5 Hz, 1H), 7.51 (d, J=16.0 Hz, 1H), 7.23 (d, J=9.2 Hz, 1H), 4.76 (t, J=7.2 Hz, 2H), 2.04 (s, 6H), 1.92 (dt, J=13.3 Hz, 2H), 1.04 (t, J=7.3Hz, 3H).

[0088] Synthesis of the fluorescent probe BH-PDN: Compound BH-PDO (250 mg, 0.47 mmol) was weighed and dissolved in 10 mL of ethyl acetate. After stirring until the solid was completely dissolved, stannous chloride dihydrate (2.134 g, 9.48 mmol) and concentrated hydrochloric acid (1.8 mL) were added and heated to 80 °C for reflux for 3.5 h. After the reaction, the reaction solution was cooled to room temperature, the ethyl acetate was removed by rotary evaporation, and 1 mL of saturated brine was added. The pH was adjusted to 8-10 with 15% sodium hydroxide solution, and the product was extracted with ethyl acetate and distilled under reduced pressure to obtain a purple crude product. The purple crude product was then purified by silica gel column chromatography (eluent: dichloromethane / methanol = 175 / 1 to 8 / 1, v / v) to obtain 149 mg of the fluorescent probe BH-PDN as a purple solid with a yield of 62%. The H NMR spectrum of the probe BH-PDN is shown below. Figure 1 As shown, 1 H NMR (400 MHz, DMSO-d6) δ 8.33 (d, J=8.5 Hz,1H), 8.27-8.10 (m, 3H), 7.96 (d, J=9.0 Hz, 1H), 7.81-7.69 (m, 2H), 7.65-7.59(m, 1H), 1.99 (s, 3H), 1.91(s, 3H), 1.87 (dd, J=7.9 Hz, 2H), 1.03 (t, J=7.4 Hz, 3H).

[0089] The 13C NMR of the fluorescent probe obtained in this example is as follows Figure 2 shown. 13 C NMR (101 MHz, DMSO-d6)δ 179.66, 172.43, 153.83, 146.68, 139.44, 136.11, 132.69, 131.08, 129.94,129.40, 127.56, 126.93, 124.41, 123.04, 114.10, 112.97, 109.76, 103.26,74.44, 52.63, 32.00, 29.89, 26.98, 21.55, 11.36.

[0090] The HRMS graph of the fluorescent probe obtained in this example is as follows: Figure 3 HRMS (ESI) calculated for C 25 H 28 IN3[MI - ] 370.2270, found 270.2286.

[0091] Example 2

[0092] The response effect of the fluorescent probe obtained in Test Example 1 on methylglyoxal was as follows:

[0093] A 10 mM stock solution of the compound BH-PDN in Example 1 was prepared using DMSO; a 100 mM stock solution of methylglyoxal was prepared using ultrapure water. 1 μL of the stock solution of the compound BH-PDN and 999 μL of PBS (pH = 7.4) buffer were placed in a cuvette and the fluorescence spectrum was measured at 625 nm. At the same time, 1 μL of the stock solution of the compound BH-PDN, 998 μL of PBS (pH = 7.4) buffer, and 1 μL of methylglyoxal solution were placed in a 37°C water bath for incubation and then transferred to a cuvette. Figure 4 As can be seen in the figure, the probe has no fluorescence. As the reaction time increases, the fluorescence intensity of the probe at 625 nm gradually increases. After 45 minutes of reaction, the fluorescence intensity reaches a plateau, indicating that the probe is a fluorescence-enhancing probe that recognizes methylglyoxal. When the reaction time reaches 45 minutes, the fluorescence intensity no longer increases, and the reaction ends.

[0094] Example 3

[0095] Fluorescence spectra of the fluorescent probe obtained in Test Example 1 reacting with methylglyoxal at different concentrations:

[0096] The solution of the above-mentioned compound BH-PDN (working concentration 10 μM), PBS (pH = 7.4) buffer, and methylglyoxal at different working concentrations (0, 1, 2, 4, 6, 8, 16, 20, 30, 40, 50, 60, 70, 80, 90, 100 μM) were added to the cuvette and reacted for 45 minutes. The fluorescence spectra at 625 nm are as follows: Figure 5 As shown, the fluorescence intensity of the reaction system at 625 nm increases with the increase of the concentration of methylglyoxal, indicating that the probe can detect methylglyoxal at different concentrations.

[0097] Example 4

[0098] The detection limit of the fluorescent probe obtained in Example 1 for detecting methylglyoxal was determined as follows:

[0099] BH-PDN solution (working concentration 10 μM), PBS (pH = 7.4) buffer and methylglyoxal at different working concentrations (0, 2, 4, 8, 12, 20, 30, 40, 50, 60, 70, 80 μM) were added to the cuvette and reacted for 45 minutes. The linear relationship between the fluorescence intensity at 625 nm and methylglyoxal is shown in the figure. Figure 6 As shown, the fluorescence intensity at 625 nm shows a good linear relationship with methylglyoxal, indicating that the fluorescent probe can quantitatively detect methylglyoxal within the range of (0-80 μM). Furthermore, based on the linear relationship plot, the detection limit of the probe for methylglyoxal was calculated to be 0.078 μM, indicating that the probe is relatively sensitive and can detect lower concentrations of methylglyoxal.

[0100] Example 5

[0101] The selectivity of the fluorescent probe obtained in Test Example 1 in recognizing methylglyoxal was as follows:

[0102] 1 μL of the fluorescent probe obtained in Example 1 (10 μM, dissolved in PBS buffer solution with a pH of 7.4 at 37°C) and a mixture of different analytes (a: free; 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), and the fluorescence intensity of each mixture at 625 nm was tested after 45 minutes of reaction. Figure 7As shown, in the presence of various anions, metal cations and amino acids, the fluorescence signal of the fluorescent probe remains basically unchanged, while after the addition of methylglyoxal, its fluorescence is significantly enhanced, indicating that the fluorescent probe has a high selectivity for methylglyoxal.

[0103] Example 6

[0104] The fluorescent probe obtained in Test Example 1 was fixed on a test strip for convenient detection and identification of methylglyoxal:

[0105] The fluorescent probe obtained in Example 1 was fixed on filter paper as a carrier by soaking and drying to make a portable detection carrier. Figure 8 The probe-loaded test paper shown here exhibited no fluorescence. However, 30 minutes after adding MGO solutions of varying concentrations, the fluorescence of the test paper gradually turned red under 365 nm UV light, and this fluorescence increased with increasing MGO concentration. This demonstrates that the device not only enables visual analysis but also meets the requirements of rapid on-site testing, especially in areas where equipment is scarce.

[0106] Example 7

[0107] The fluorescent probe (10 μM) obtained in Example 1 was co-incubated with A546 cells, and the cells were washed with PBS and then imaged under a microscope. The first group was the control group, which was incubated with the fluorescent probe for 30 min. The second group was the curcumin group, which was incubated with curcumin solutions of different concentrations (50, 100, 200 μM) and then incubated with the fluorescent probe. The third group was the curcumin + N-acetylcysteine ​​group. N-acetylcysteine ​​(NAC) is a common MGO scavenger that can clear MGO in cells. Incubated with 200 μM curcumin, then incubated with 200 μM N-acetylcysteine, and finally incubated with the fluorescent probe. The results are as follows. Figure 9 As shown, the fluorescence intensity after incubation with curcumin was higher than that of the control group, and the fluorescence increased with increasing curcumin concentration. After incubation with N-acetylcysteine, the fluorescence intensity decreased. This indicates that the probe can detect changes in MGO content in cells regulated by curcumin and N-acetylcysteine.

[0108] Example 8

[0109] The fluorescent probe obtained in Example 1 was used to detect methylglyoxal in living zebrafish.

[0110] Zebrafish fry grown for 0-7 days were cultured in 6-well plates and divided into three groups. The first group was the control group, which was incubated with a fluorescent probe for 30 minutes. The second group was the curcumin group, which was incubated with curcumin solutions of different concentrations (50, 100, 200 μM) and then incubated with a fluorescent probe. The third group was the curcumin + N-acetylcysteine ​​group. It was incubated with 200 μM curcumin, then incubated with 200 μM N-acetylcysteine, and finally incubated with a fluorescent probe. Figure 10 As shown in the figure, as the concentration of curcumin increases, the fluorescence intensity of the zebrafish becomes stronger, while after pretreatment with the scavenger, the zebrafish has almost no fluorescence, indicating that the probe can specifically detect the activity of endogenous methylglyoxal in living organisms.

[0111] Example 9

[0112] The fluorescent probe obtained in Example 1 was used to detect imaging of methylglyoxal in the root tip tissue of Arabidopsis thaliana.

[0113] The Arabidopsis thaliana culture grown for 7 days was divided into three groups. The first group was the control group, which was incubated with the fluorescent probe for 30 minutes. The second group was the methylglyoxal group, which was incubated with 100 μM methylglyoxal solution and then incubated with the fluorescent probe. The third group was the methylglyoxal + N-acetylcysteine ​​group. It was incubated with 100 μM methylglyoxal, then incubated with 200 μM N-acetylcysteine, and finally incubated with the fluorescent probe. Figure 11 As shown, with the addition of methylglyoxal, the fluorescence intensity of the root tip tissue of Arabidopsis thaliana becomes stronger, while after pretreatment with the scavenger, Arabidopsis thaliana has almost no fluorescence, indicating that the probe can quantitatively analyze the content of methylglyoxal in plants.

[0114] Other beneficial effects of the present invention include:

[0115] (1) The fluorescent probe prepared by the present invention has low fluorescence background, good chemical stability, good water solubility, high sensitivity (0.078 μM), and high selectivity;

[0116] (2) The fluorescent probe prepared by the present invention has a large Stokes shift (255 nm) and strong anti-interference ability, and can specifically detect methylglyoxal;

[0117] (3) The fluorescent probe prepared by the present invention can be loaded on a test strip for convenient detection;

[0118] (4) The probe has the advantages of good water solubility, large Stokes shift, and strong anti-interference ability. It can not only detect methylglyoxal in living cells, but also detect endogenous methylglyoxal in zebrafish and Arabidopsis.

[0119] The specific embodiments of the present invention described above do not limit the scope of protection of the present invention. Any other corresponding changes and modifications made based on the technical concept of the present invention should be included in the scope of protection of the claims of the present invention.

Claims

1. A fluorescent probe for detecting methylglyoxal using a large Stokes shift, characterized in that: Its structural formula is shown in formula (I): 。 2. The method for preparing a fluorescent probe according to claim 1, wherein The following steps are involved: S1. Dissolve compound C-3 in a first solvent, then add concentrated hydrochloric acid and heat to 75-85° C. for reflux reaction to obtain the intermediate BH-PDO; S2, dissolving the intermediate BH-PDO in a second solvent, then adding stannous chloride dihydrate and concentrated hydrochloric acid, heating to 75-85° C. for reflux reaction to obtain the fluorescent probe; The structural formula of the compound C-3 is as follows: ； The structural formula of the intermediate BH-PDO is as follows: 。 3. The method for preparing a fluorescent probe according to claim 2, wherein: In step S1, the first solvent is ethyl acetate; in step S2, the second solvent is ethyl acetate; in step S1, the reflux reaction is carried out for 4-8 hours; in step S2, the reflux reaction is carried out for 4-8 hours.

4. The method for preparing a fluorescent probe according to claim 2, wherein: In step S1, the compound C-3 is prepared by the following steps: Intermediate C-2 and intermediate D-2 are dissolved in a third solvent, followed by addition of piperidine, and the mixture is refluxed at 75-85°C to obtain intermediate C-3; The structural formula of the intermediate C-2 is: The structural formula of the compound D-2 is: The intermediate C-2 is prepared by the following steps: dissolving compound C-1 in a fourth solvent and adding 1-iodopropane, and reacting under reflux at 75-85°C to obtain the intermediate C-2; The structural formula of the compound C-1 is: ; The intermediate D-2 is prepared by the following steps: Mix concentrated nitric acid and concentrated sulfuric acid to obtain a mixed solution, and then slowly add the mixed solution dropwise to the reaction vessel containing compound D-1 to react and obtain the intermediate D-2; The structural formula of the compound D-1 is: .

5. The method for preparing a fluorescent probe according to claim 4, wherein: The third solvent is anhydrous acetonitrile; and the fourth solvent is anhydrous acetonitrile.

6. Use of the fluorescent probe according to claim 1 or the fluorescent probe prepared by the preparation method according to any one of claims 2 to 5 in detecting methylglyoxal in an environment or a biological sample, wherein the detection of methylglyoxal in the biological sample is for a non-disease diagnosis purpose.

7. The use according to claim 6, characterized in that The product of the fluorescent probe (I) reacting with methylglyoxal has a maximum emission wavelength at 625 nm and exhibits red fluorescence.

8. The use according to claim 7, characterized in that The structural formula of the product is shown in formula (II): 。 9. The use according to claim 6, characterized in that Qualitative and quantitative detection of methylglyoxal in aqueous solution; or, imaging detection in cells, zebrafish or Arabidopsis; the imaging detection of cells and zebrafish is for non-disease diagnosis purposes.

10. A fluorescent reagent detection kit, characterized in that: The invention comprises the fluorescent probe according to claim 1 or the fluorescent probe prepared by the preparation method according to any one of claims 2 to 5.

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