Fluorescent probe as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine detection, and provides a fluorescent probe as well as a preparation method and application thereof. The fluorescent probe disclosed by the invention can emit relatively weak fluorescence and also can specifically and rapidly react with the ALDH2 enzyme to generate a product with strong fluorescence, and the fluorescence intensity is positively correlated with the concentration of the ALDH2 enzyme. The fluorescent probe has the beneficial effects that (1) the fluorescent probe used for detecting the enzyme activity of ALDH2 is good in molecular stability and can be stored and used for a long time; and (2) the fluorescent signal brightness is high, the detection signal-to-noise ratio is high, and the sensitivity is good. (3) the selectivity is excellent, and ALDH2 enzyme can be specifically detected in a complex biological sample. And (4) good biological membrane permeability is achieved, so that the method can be used for detecting the enzyme activity of ALDH2 in living cells. And (5) the detection speed is high. And (6) the detection response is sensitive, and the signal intensity can be enhanced along with the increase of the ALDH2 enzyme content in the species to be detected.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, and in particular to a fluorescent probe, its preparation method, and its application. Background Technology

[0002] Aldehydes are relatively reactive organic compounds characterized by polarized carbon-oxygen double bonds. They can be classified into endogenous and exogenous types and possess strong carcinogenic and cytotoxic properties. Environmentally derived aldehydes, such as formaldehyde, acrolein, acetaldehyde, and crotonaldehyde, can interact with endogenous aldehydes produced during oxidative stress, such as malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), thus accelerating disease progression. The accumulation of cytotoxic aldehydes in the body can impair the function of biological macromolecules (such as proteins, DNA, and RNA), inducing a range of related diseases, including alcoholism, osteoporosis, Fanconi anemia, pain, diabetic complications, neurodegenerative diseases, cardiovascular diseases, and cancer.

[0003] Aldehyde dehydrogenases (ALDHs) are a class of enzymes that use various aldehydes as substrates, primarily catalyzing the oxidation of acetaldehyde and other aliphatic aldehydes. They have attracted widespread attention as therapeutic targets for many related diseases. Currently, 19 human ALDHs are known. ALDHs mainly work by oxidizing NAD(P)... + It is reduced to NAD(P)H, catalyzing the oxidation of acetaldehyde and other aliphatic aldehydes to form their corresponding acids. Given the important role of ALDHs in maintaining aldehyde homeostasis, they have become potential targets for the treatment of toxic aldehyde-related diseases.

[0004] ALDH2 is a tetrameric protein with three domains: a coenzyme-binding domain, a catalytic domain, and an oligomeric domain. While transcription and translation of ALDH2 can be quantified using methods such as quantitative real-time PCR (qPCR) and Western blotting (WB), these traditional immunorecognition-based methods only determine protein expression levels and do not reflect its functional state. Since enzyme function is ultimately determined by activity, not concentration, monitoring enzyme activity is crucial. Measuring the characteristic UV absorbance of the metabolite NADH at 340 nm to quantify ALDH enzyme activity is an indirect method, but it suffers from drawbacks such as time consumption, poor sensitivity, and non-in situ imaging.

[0005] Fluorescent probes have attracted widespread attention due to their high sensitivity and ability to directly evaluate enzyme activity in the native cell environment. This method not only overcomes difficulties such as protein purification but also provides a biocompatible detection method for in-situ imaging and direct enzyme activity reading. Currently, the commercially available kit for ALDH enzyme activity detection is ALDEFLUOR. TMThe working principle is to use ALDH to convert the uncharged substrate BAAA, which can diffuse into living cells, into the negatively charged product BAA. - The product remains intracellular and emits fluorescence. Therefore, cells highly expressing ALDH will exhibit bright fluorescence. However, the fluorescence "on" state does not change during substrate oxidation, resulting in high background fluorescence, requiring multiple washes and easily causing false positive results. Furthermore, ALDEFLUOR... TM As a pan-ALDH activity assay kit, it also lacks subtype specificity. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide a fluorescent probe, its preparation method, and its application. The fluorescent probe provided by this invention exhibits high specificity and sensitivity when used for ALDH2 enzyme activity detection.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a fluorescent probe having the structure shown in Formula I:

[0009]

[0010] In Equation I, R is

[0011] R1 can be hydroxyl, amino, substituted amino, cycloalkylamine, or heterocycloalkylamine.

[0012] Preferably, the substituted amino group is an amino group in which two hydrogen atoms are replaced by an alkyl group, and the alkyl group is a straight-chain alkyl group with 1 to 6 carbon atoms.

[0013] Preferably, the cycloalkylamine group is

[0014] Preferably, the heterocyclic alkylamine group is

[0015] Preferably, the specific structure is as follows:

[0016]

[0017]

[0018] The present invention also provides a method for preparing the fluorescent probe described in the above technical solution, comprising the following steps:

[0019] The precursor compound shown in Formula 2, ((1,3-dioxacyclopentan-2-yl)methyl)triphenylphosphonium bromide, 18-crown ether-6, and potassium carbonate were mixed and subjected to a first reaction to obtain an intermediate;

[0020] The intermediate was deprotected to obtain the fluorescent probe;

[0021] R-CHO Formula 2.

[0022] Preferably, the molar ratio of the precursor compound to ((1,3-dioxane-2-yl)methyl)triphenylphosphonium bromide is 1:1 to 1:2, the molar ratio of the precursor compound to 18-crown ether-6 is 1:1 to 1:2, and the molar ratio of the precursor compound to potassium carbonate is 1:1 to 1:2; the first reaction is carried out under nitrogen protection and reflux; the reaction time is 12 to 24 hours.

[0023] Preferably, the deprotection reagent is a hydrochloric acid solution with a concentration of 0.5–1.5 mol / L; the deprotection temperature is an ice-water bath, and the time is 15–60 min.

[0024] This invention also provides the application of the fluorescent probe described in the above technical solution in the preparation of reagents for detecting ALDH2 enzyme activity.

[0025] Preferably, the reagent for detecting ALDH2 enzyme activity is used in the form of test strips, probe solutions, or kits.

[0026] This invention provides a fluorescent probe.

[0027] The fluorescent probe of this invention emits weak fluorescence but reacts specifically and rapidly with the ALDH2 enzyme to generate a product with strong fluorescence, and the fluorescence intensity is positively correlated with the ALDH2 enzyme concentration. The fluorescent probe of this invention is suitable for detecting ALDH2 enzyme activity in solution, intracellularly, or in vivo tissue.

[0028] The fluorescent probe provided by this invention has the following beneficial effects: (1) As a fluorescent probe for detecting ALDH2 enzyme activity, it has good molecular stability and can be stored and used for a long time. (2) It has high fluorescence signal brightness, high detection signal-to-noise ratio, and good sensitivity. (3) It has excellent selectivity and can specifically detect ALDH2 enzyme in complex biological samples. (4) It has good biomembrane permeability, and therefore can be used for the detection of ALDH2 enzyme activity in living cells. (5) It has a fast detection speed. (6) It has a sensitive detection response, and the signal intensity increases with the increase of ALDH2 enzyme content in the test species. Attached Figure Description

[0029] Figure 1The fluorescence intensity diagrams at the maximum emission wavelengths are obtained for fluorescent probe I-9 and its response blank, ALDH2, superoxide anion, hydrogen peroxide, singlet oxygen, sodium hypochlorite, tert-butyl hydrogen peroxide, peroxynitrite anion, nitric oxide, hydroxyl radical, reduced glutathione, cysteine, homocysteine, magnesium ion, calcium ion, ferric ion, ferrous ion, potassium ion, and zinc ion.

[0030] Figure 2 The fluorescence intensity diagram of fluorescent probe I-9 and its response to ALDH1A1, ALDH1A3, ALDH3A1, and ALDH4A1 at the maximum emission wavelength is shown.

[0031] Figure 3 The fluorescence intensity of fluorescent probe I-9 and its response to pancreatic enzymes, lipases, chymotrypsin, antioxidant enzymes, esterases and β-galactosidase at the maximum emission wavelength is shown.

[0032] Figure 4 The graph shows the changes in fluorescence intensity of the fluorescent probe I-9 in the presence of different concentrations of ALDH2.

[0033] Figure 5 The graph shows the changes in fluorescence intensity of different concentrations of I-9 compound in the presence of the same concentration of ALDH2.

[0034] Figure 6 Image of BV2 cells after pretreatment with different concentrations of agonist and inhibitor using fluorescent probe I-9;

[0035] Figure 7 The fluorescence intensity of BV2 cells after pretreatment with different concentrations of agonist and inhibitor using fluorescent probe I-9;

[0036] Figure 8 The results show the enzyme activity of fluorescent probe I-9 in tissue homogenates from 6-month-old mice. Detailed Implementation

[0037] This invention provides a fluorescent probe having the structure shown in Formula I:

[0038]

[0039] In Equation I, R is

[0040]

[0041] R1 can be hydroxyl, amino, substituted amino, cycloalkylamine, or heterocycloalkylamine.

[0042] In this invention, the substituted amino group is preferably one in which two hydrogen atoms on the amino group are replaced by an alkyl group, and the alkyl group is preferably a straight-chain alkyl group with 1 to 6 carbon atoms, and more preferably a straight-chain alkyl group with 1 to 2 carbon atoms.

[0043] In this invention, the cycloalkanamine group can be...

[0044] In this invention, the heterocyclic alkylamine group can be...

[0045] In this invention, the specific structure of the fluorescent probe can be as follows:

[0046]

[0047] The present invention also provides a method for preparing the fluorescent probe described in the above technical solution, comprising the following steps:

[0048] The precursor compound shown in Formula 2, ((1,3-dioxacyclopentan-2-yl)methyl)triphenylphosphonium bromide, 18-crown ether-6, and potassium carbonate were subjected to a first reaction to give an intermediate;

[0049] The intermediate was deprotected to obtain the fluorescent probe;

[0050] R-CHO Formula 2.

[0051] Unless otherwise specified, all raw materials used in this invention are preferably commercially available products.

[0052] In this invention, the precursor compound shown in Formula 2, ((1,3-dioxacyclopentan-2-yl)methyl)triphenylphosphonium bromide, 18-crown ether-6 and potassium carbonate are mixed and subjected to a first reaction to obtain an intermediate.

[0053] In this invention, when R1 in the R substituent of Formula 2 is a hydroxyl group, the precursor compound shown in Formula 2 can be purchased directly.

[0054] In this invention, when R1 in the substituent R in Formula 2 is not a hydroxyl group, that is, when R1 in the substituent R is an amino group, a substituted amino group, a cycloalkylamine group, or a heterocycloalkylamine group, the preparation method of the precursor compound preferably includes the following steps: under the action of p-toluenesulfonic acid pyridine salt, the compound shown in Formula 3 undergoes an acetal reaction with ethylene glycol to obtain an acetal product; the acetal product is coupled with R1-H under the conditions of cesium carbonate, palladium acetate, and 1,1'-binaphthyl-2,2'-bisdiphenylphosphine to undergo a coupling reaction, and then the acetal protection is removed under acidic conditions to obtain the precursor compound;

[0055] The compound shown in Formula 3 has the following structure:

[0056]

[0057] In this invention, the medium for the acetal reaction is preferably toluene. In this invention, the molar ratio of the compound shown in Formula 3, ethylene glycol, and p-toluenesulfonic acid pyridinium salt is preferably 1:2:0.1. In this invention, the acetal reaction is preferably carried out under reflux and water separation conditions. After the acetal reaction, this invention preferably further includes post-treatment, which preferably includes: cooling, quenching the acetal reaction with water, extraction with ethyl acetate, and sequentially washing the obtained organic phase with water, drying with anhydrous sodium sulfate, and concentrating to obtain the acetal product.

[0058] In this invention, the coupling reaction medium is preferably toluene. In this invention, the molar ratio of the acetal product, R1-H, cesium carbonate, palladium acetate, and 1,1'-binaphthyl-2,2'-bis(diphenylphosphine) is preferably 1:1:0.1:0.1:0.1. In this invention, the coupling reaction temperature is preferably 110°C, and the reaction time is preferably 12 h. After the coupling reaction, this invention preferably includes post-treatment, which preferably includes: quenching the reaction with water, extraction with ethyl acetate, and sequentially washing the obtained organic phase with water, drying with anhydrous sodium sulfate, concentration, and purification by silica gel column chromatography to obtain the coupling product. In this invention, the eluent for the silica gel column chromatography purification is preferably a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 4:1.

[0059] In this invention, the reagent for acetal removal protection is preferably a hydrochloric acid solution, and the mass fraction of the hydrochloric acid solution is preferably 2-4%. In this invention, the temperature for acetal removal protection is preferably room temperature, and the time is preferably 1-2 hours. After acetal removal protection, this invention preferably further includes post-treatment, which preferably includes: extracting the acetal removal protection solution with ethyl acetate, and then sequentially washing the obtained organic phase with water, drying it with anhydrous sodium sulfate, and concentrating it to obtain the precursor compound.

[0060] In this invention, the medium for the first reaction is preferably toluene.

[0061] In this invention, the molar ratio of the precursor compound to ((1,3-dioxacyclopentan-2-yl)methyl)triphenylphosphonium bromide is preferably 1:1 to 1:2, specifically 1:1, 1:1.5, or 1:2. In this invention, the molar ratio of the precursor compound to 18-crown ether-6 is preferably 1:1 to 1:2, specifically 1:1, 1:1.5, or 1:2. In this invention, the molar ratio of the precursor compound to potassium carbonate is preferably 1:1 to 1:2, specifically 1:1, 1:1.5, or 1:2.

[0062] In this invention, the first reaction is preferably carried out under nitrogen protection and heating under reflux conditions; the reaction time is preferably 12 to 24 hours, specifically 12 hours, 18 hours or 24 hours.

[0063] Following the first reaction, the present invention preferably further includes: mixing the obtained first reaction solution with an organic extractant, extracting to obtain an organic phase, and sequentially washing, drying, rotary evaporating, purifying by column chromatography, and distilling under reduced pressure to obtain an intermediate. In this invention, the organic extractant is preferably ethyl acetate. In this invention, the washing preferably includes sequential washing with water and washing with saturated brine, and the number of times each washing is performed is preferably three. In this invention, the drying is preferably drying with a desiccant, and the desiccant is preferably anhydrous sodium sulfate. The present invention does not specifically limit the rotary evaporation operation; any rotary evaporation operation well known to those skilled in the art can be used. In this invention, the reagent for column chromatography purification is preferably a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 20:1. The present invention does not specifically limit the operation of reduced pressure distillation, as long as the solvent can be removed.

[0064] After obtaining the intermediate, the present invention deprotects the intermediate to obtain the fluorescent probe.

[0065] In this invention, the intermediate is preferably dissolved before deprotection, and the dissolving agent is preferably tetrahydrofuran.

[0066] In this invention, the deprotecting reagent is preferably a hydrochloric acid solution, and the concentration of the hydrochloric acid solution is preferably 0.5–1.5 mol / L, specifically 0.5 mol / L, 1 mol / L, or 1.5 mol / L. In this invention, the deprotecting reagent is preferably added dropwise to the intermediate, and the addition temperature is preferably an ice-water bath.

[0067] In this invention, the temperature for deprotection is preferably an ice-water bath, and the time is preferably 15 to 60 minutes, specifically 15 minutes, 30 minutes, 45 minutes, or 60 minutes.

[0068] After deprotection, the present invention preferably further includes: adjusting the pH of the obtained deprotected solution to neutral, then adding ethyl acetate for extraction to obtain an ethyl acetate phase; washing, drying, and rotary evaporating the ethyl acetate phase to obtain the fluorescent probe. In this invention, the reagent used to adjust the pH of the obtained deprotected solution to neutral is preferably a saturated NaHCO3 solution. In this invention, the washing preferably includes sequential washing with water and washing with saturated brine, and the number of times each washing is performed is preferably three. In this invention, the drying method is preferably drying with a desiccant, and the desiccant is preferably anhydrous sodium sulfate. The present invention does not specifically limit the rotary evaporation operation; any operation well known to those skilled in the art can be used.

[0069] This invention also provides the application of the fluorescent probe described in the above technical solution in the preparation of reagents for detecting ALDH2 enzyme activity.

[0070] In this invention, the ALDH2 enzyme can be a recombinant protein, intracellular or living tissue ALDH2.

[0071] In this invention, the reagent for detecting ALDH2 enzyme activity is preferably used in the form of test strips, probe solutions, or kits.

[0072] The following detailed description of the fluorescent probes, their preparation methods, and applications provided by the present invention, with reference to specific embodiments, should not be construed as limiting the scope of protection of the present invention.

[0073] Example 1

[0074] Synthesis of fluorescent probe I-9:

[0075]

[0076] The precursor compound (1 eq), 18-crown ether-6 (0.02 eq), ((1,3-dioxacyclopentan-2-yl)methyl)triphenylphosphonium bromide (1.5 eq), and K₂CO₃ (1.5 eq) were dissolved in 7 mL of toluene. The reaction mixture was refluxed under nitrogen protection for 18 h. After the reaction was complete, ethyl acetate was added to extract the organic phase, which was then washed three times each with water and saturated brine. The washed organic phase was dried over anhydrous sodium sulfate, evaporated to dryness, and purified by silica gel column chromatography using a 20:1 (v / v) mixture of petroleum ether and ethyl acetate as eluent. The solvent was removed by vacuum distillation to obtain the intermediate.

[0077] The intermediate was dissolved in 20 mL of tetrahydrofuran (THF), and 8 mL of 1 mol / L HCl was slowly added dropwise under ice bath with stirring for 30 min. The pH of the reaction mixture was adjusted to 7.0 with saturated NaHCO3 solution, and the organic phase was extracted with ethyl acetate. The organic phase was washed three times each with water and saturated brine, and dried over anhydrous sodium sulfate. The dried phase was then evaporated to dryness to give I-9 (yield 47%). ESI-MS (m / z): 281 [M+H]. + .

[0078] The methods for preparing the precursor compounds include:

[0079] Commercially available 6-bromo-2-aldehyde naphthalene was reacted with ethylene glycol in toluene under reflux to remove water, with p-toluenesulfonic acid pyridinium salt added as a catalyst. The molar ratio of 6-bromo-2-aldehyde naphthalene, ethylene glycol, and p-toluenesulfonic acid pyridinium salt was 1:2:0.1. The reaction was carried out for 12 h. After cooling, the reaction solution was quenched with water and extracted with ethyl acetate. The obtained ethyl acetate phase was washed with water, dried over anhydrous sodium sulfate, and concentrated to obtain an acetal product. The acetal product was then coupled with N-methylpiperazine in toluene in the presence of cesium carbonate, palladium acetate, and 1,1'-binaphthyl-2,2'-bis(diphenylphosphine). The acetal product, N-methylpiperazine, cesium carbonate, palladium acetate, and... The molar ratio of 1,1'-binaphthyl-2,2'-bis(diphenylphosphine) was 1:1:0.1:0.1:0.1, the reaction temperature was 110℃, and the reaction time was 12 h. Post-treatment involved quenching the reaction with water, extraction with ethyl acetate, and subsequent washing, drying with anhydrous sodium sulfate, concentration, and silica gel column chromatography purification. The eluent for silica gel column chromatography was a mixture of petroleum ether and ethyl acetate in a volume ratio of 4:1. Then, 3% hydrochloric acid solution was added for deprotection for 1 h. The deacetalized solution was extracted with ethyl acetate, and the resulting ethyl acetate phase was subsequently washed with water, dried with anhydrous sodium sulfate, and concentrated to obtain the precursor compound.

[0080] Example 2

[0081] I-1 (yield 94%), ESI-MS (m / z): 199 [M+H] + .

[0082] Example 3

[0083] I-2 (yield 62%), ESI-MS (m / z): 198 [M+H] + .

[0084] Example 4

[0085] I-3 (yield 57%), ESI-MS (m / z): 226 [M+H] + .

[0086] Example 5

[0087] I-4 (yield 56%), ESI-MS (m / z): 254 [M+H] + .

[0088] Example 6

[0089] I-5 (yield 50%), ESI-MS (m / z): 252 [M+H] + .

[0090] Example 7

[0091] I-6 (yield 42%), ESI-MS (m / z): 266 [M+H] + .

[0092] Example 8

[0093] I-7 (yield 79%), ESI-MS (m / z): 268 [M+H] + .

[0094] Example 9

[0095] I-8 (yield 32%), ESI-MS (m / z): 267 [M+H] + .

[0096] Example 10

[0097] I-10 (yield 38%), ESI-MS (m / z): 200 [M+H] + .

[0098] Example 11

[0099] I-11 (yield 45%), ESI-MS (m / z): 199 [M+H] + .

[0100] Example 12

[0101] I-12 (yield 65%), ESI-MS (m / z): 227 [M+H] + .

[0102] Example 13

[0103] I-13 (yield 69%), ESI-MS (m / z): 255 [M+H] + .

[0104] Example 14

[0105] I-14 (yield 78%), ESI-MS (m / z): 253 [M+H] + .

[0106] Example 15

[0107] I-15 (yield 82%), ESI-MS (m / z): 267 [M+H] + .

[0108] Example 16

[0109] I-16 (yield 80%), ESI-MS (m / z): 269 [M+H] + .

[0110] Example 17

[0111] I-17 (yield 40%), ESI-MS (m / z): 268 [M+H] + .

[0112] Example 18

[0113] I-18 (yield 50%), ESI-MS (m / z): 282 [M+H] + .

[0114] Example 19

[0115] I-19 (yield 68%), ESI-MS (m / z): 217 [M+H] + .

[0116] Example 20

[0117] I-20 (yield 60%), ESI-MS (m / z): 216 [M+H] + .

[0118] Example 21

[0119] I-21 (yield 73%), ESI-MS (m / z): 244 [M+H] + .

[0120] Example 22

[0121] I-22 (yield 59%), ESI-MS (m / z): 272 [M+H] + .

[0122] Example 23

[0123] I-23 (yield 76%), ESI-MS (m / z): 270 [M+H] + .

[0124] Example 24

[0125] I-24 (yield 73%), ESI-MS (m / z): 284 [M+H] + .

[0126] Example 25

[0127] I-25 (yield 68%), ESI-MS (m / z): 286 [M+H] + .

[0128] Example 26

[0129] I-26 (yield 35%), ESI-MS (m / z): 285 [M+H] + .

[0130] Example 27

[0131] I-27 (yield 70%), ESI-MS (m / z): 299 [M+H] + .

[0132] Test Example 1

[0133] The responsiveness of compound I-9 to ALDH2

[0134] 1 mg of the above fluorescent probe molecules were dissolved in a small amount of dimethyl sulfoxide (DMSO) to prepare a 5 mmol / L probe stock solution. Control group: The probe stock solution was diluted with PBS to a final concentration of 5 μM, and its fluorescence intensity at the maximum emission wavelength was recorded. Experimental group: Another probe stock solution was diluted with PBS to a final probe concentration of 5 μM, and reacted with 90 nmol / LALDH2 for 30 min, and its fluorescence intensity at the maximum emission wavelength was recorded. Comparing the fluorescence intensity at the maximum wavelength between the control and experimental groups, the fluorescent probe showed a significant enhancement in fluorescence to ALDH2. The experimental results are shown in Table 1.

[0135] Table 1. Fluorescence enhancement test results of probe molecules

[0136]

[0137]

[0138]

[0139] Test Example 2

[0140] Species-specific fluorescence response of fluorescent probe I-9 to ALDH2

[0141] Fluorescent probe I-9 (1 mg) was used as the probe molecule and dissolved in a small amount of dimethyl sulfoxide (DMSO) to prepare a 10 mM probe stock solution. The probe stock solution was divided into several portions, and the fluorescence intensity of one portion at the maximum emission wavelength was recorded. Several other portions were taken and the analytes were added to them respectively. The analytes included: (1) Blank; (2) ALDH2 (90 nM); (3) superoxide anion (O2· - (100μM); (4)H2O2 (10mM); (5)Singlet oxygen ( 1 (6) NaClO (20 μM); (7) tert-butyl hydroperoxide (TBHP) (100 μM); (8) peroxynitrite anion (ONOO) - )(10μM); (9)NO(20μM); (10)OH - (100 μM); (11) Reduced glutathione (GSH) (100 μM); (12) Cysteine ​​(Cys) (100 μM); (13) Homocysteine ​​(Hcy) (100 μM); (14) MgCl2 (100 μM); (15) CaCl2 (1 mM); (16) FeCl3 (100 μM); (17) FeSO4·7H2O (100 μM); (18) KCl (10 mM); (19) ZnCl2 (1 mM). After reacting for 15 min, I-9 was added, and the solution was diluted with PBS to a final probe concentration of 1 μM. Then, NAD was added using a multi-channel pipette. + The reaction was triggered at a final concentration of 2.5 mM. The fluorescence intensity changes at the maximum emission wavelength were recorded, and the results are as follows: Figure 1 As shown, Figure 1 The fluorescence intensity diagram at the maximum emission wavelength is obtained for fluorescent probe I-9 and its response blank, ALDH2, superoxide anion, hydrogen peroxide, singlet oxygen, sodium hypochlorite, tert-butyl hydroperoxide, peroxynitrite anion, nitric oxide, hydroxyl radical, reduced glutathione, cysteine, homocysteine, magnesium ion, calcium ion, ferric ion, ferrous ion, potassium ion, and zinc ion. Figure 1 It can be seen that the fluorescent probe I-9 showed a significant enhancement of fluorescence in response to ALDH2, and this change could not be caused by other species, indicating that this type of probe has a species-specific fluorescent response to ALDH2.

[0142] Test Example 3

[0143] The fluorescent probe I-9 exhibits a subtype-specific fluorescence response to ALDH2.

[0144] Fluorescent probe I-9 (1 mg) was dissolved in a small amount of dimethyl sulfoxide (DMSO) to prepare a 10 mM probe stock solution, which was then serially diluted to 0.25, 0.5, 1, 2, and 4 μmol / L. The solution was divided into several portions, and the fluorescence intensity of one portion at the maximum emission wavelength was recorded. Several other portions were then treated with equal concentrations (90 nM) of ALDH1A1, ALDH1A3, ALDH3A1, and ALDH4A1, respectively. Finally, NAD+ was added using a multi-channel pipette. + The reaction was triggered at a final concentration of 2.5 mM, and the fluorescence intensity changes at the maximum emission wavelength were recorded. The results are as follows: Figure 2 As shown, Figure 2 The image shows the fluorescence intensity of fluorescent probe I-9 and its responses to ALDH1A1, ALDH1A3, ALDH3A1, and ALDH4A1 at the maximum emission wavelength. Figure 2 It can be seen that fluorescent probe I-9 shows a significant enhancement in fluorescence for ALDH2, and other subtypes do not cause this change, indicating that this type of probe has a subtype-specific fluorescent response to ALDH2.

[0145] Test Example 4

[0146] Enzyme-specific fluorescence response of fluorescent probe I-9 to ALDH2

[0147] Dissolve 1 mg of fluorescent probe I-9 in a small amount of dimethyl sulfoxide (DMSO) to prepare a 10 mM stock solution. Divide the stock solution into several portions, and record the fluorescence intensity of one portion at its maximum emission wavelength. Take several other portions and add the same concentration (90 nM) of trypsin, lipase, chymotrypsin, antioxidant enzyme, esterase, and β-galactosidase to each. Then add I-9, dilute with PBS to a final probe concentration of 10 μM, and then add NAD+ using a multi-channel pipette. + The reaction was triggered at a final concentration of 2.5 mM. The fluorescence intensity changes at the maximum emission wavelength were recorded, and the results are as follows: Figure 3 , Figure 3 This is a fluorescence intensity diagram of fluorescent probe I-9 and its response to trypsin, lipase, chymotrypsin, antioxidant enzyme, esterase, and β-galactosidase at the maximum emission wavelength. Figure 3 It can be seen that fluorescent probe I-9 shows a significant enhancement in fluorescence for ALDH2, and other common proteins cannot cause this change, indicating that this type of probe has a common protein-specific fluorescent response to ALDH2.

[0148] Test Example 5

[0149] Changes in fluorescence intensity of fluorescent probe I-9 at different concentrations of ALDH2

[0150] Fluorescent probe I-9 (1 mg) was dissolved in a small amount of dimethyl sulfoxide (DMSO) to prepare a 10 mM stock solution. The stock solution was divided into several portions, and the fluorescence intensity of one portion at the maximum emission wavelength was recorded. Several other portions were prepared, and PBS buffer containing different concentrations of ALDH2 (0, 5.625, 11.25, 22.5, 45, and 90 nM) was added. I-9 was then added, and the solution was diluted with PBS to a final probe concentration of 10 μM. NAD+ was then added using a multi-channel pipette. + The reaction was triggered at a final concentration of 2.5 mM. The fluorescence intensity changes at the maximum emission wavelength were recorded, and the results are as follows: Figure 4 As shown, Figure 4 This is a graph showing the fluorescence intensity changes of fluorescent probe I-9 in the presence of different concentrations of ALDH2. Figure 4 It can be seen that the fluorescence intensity of fluorescent probe I-9 increases significantly with the increase of ALDH2 concentration, indicating that this type of probe has good sensitivity to ALDH2.

[0151] Test Example 6

[0152] Changes in fluorescence intensity of different concentrations of fluorescent probe I-9 in ALDH2

[0153] Fluorescent probe I-9 (1 mg) was dissolved in a small amount of dimethyl sulfoxide (DMSO) to prepare a 10 mM stock solution. The stock solution was divided into several portions, and the fluorescence intensity of one portion at the maximum emission wavelength was recorded. Several other portions were prepared, and ALDH2 (90 nM) in PBS buffer was added, followed by I-9. The solutions were diluted with PBS to final probe concentrations of 0, 0.25 μM, 0.5 μM, 1 μM, 2 μM, and 4 μM. NAD+ was then added using a multi-channel pipette. + The reaction was triggered at a final concentration of 2.5 mM. The fluorescence intensity changes at the maximum emission wavelength were recorded, and the results are as follows: Figure 5 As shown, from Figure 5 It can be seen that, at the same ALDH2 concentration, the fluorescence intensity of the system increases with the increase of I-9 concentration, further illustrating the dependence of probe fluorescence intensity on ALDH2.

[0154] Test Example 7

[0155] Detection of ALDH2 enzyme activity by fluorescent probe I-9 on different concentrations of regulators in BV2, HepG2, and SH-SY5Y cells

[0156] Fluorescent probe I-9 (1 mg) was dissolved in a small amount of dimethyl sulfoxide (DMSO) to prepare a probe solution. Cells were pre-incubated with different concentrations of the agonist alda-1 and the inhibitor disulfiram (0, 2.5, 5, 10 μM) for 4 h. The probe was then diluted to 10 μM with culture medium and added to BV2 cells. After incubation for 30 min, imaging was performed using a fluorescence microscope. The results are shown below. Figure 6 and Figure 7 As shown, Figure 6 Images of BV2 cells after pretreatment with different concentrations of agonist and inhibitor using the fluorescent probe I-9. Figure 7 The image shows the fluorescence intensity of BV2 cells after pretreatment with different concentrations of agonist and inhibitor using the fluorescent probe I-9. Figure 6 and Figure 7 It can be seen that the fluorescence intensity increases with increasing agonist concentration and decreases with decreasing agonist concentration. This indicates that the fluorescent probe I-9 can detect changes in ALDH2 enzyme activity in live cells.

[0157] Test Example 8

[0158] Detection of ALDH2 enzyme activity in mouse tissue homogenate using fluorescent probe I-9

[0159] Three 6-month-old male black mice (C57BL / 6JJcl) were euthanized after blood was collected from their orbital fossa. Tissue samples (heart, liver, spleen, lung, kidney, and brain) were immediately collected. The tissues were rinsed with cold PBS to remove any remaining blood and contents, blotted dry, labeled, and divided into two portions. Each portion was weighed and placed in a 1.5 mL cryovial. The tissues were immediately freeze-dried in liquid nitrogen at -80°C. The freeze-dried tissues were homogenized, and a homogenate was prepared by adding twice the volume of phosphate-buffered saline (pH 7.4). The homogenate was placed in a 1.5 mL plastic tube and centrifuged (3000 rpm × 10 min, 4°C). The supernatant was collected and stored as a tissue homogenate at -80°C.

[0160] A fixed volume of supernatant was added to a PBS buffer (10 mM, pH 7.4), and fluorescent probe I-9 (final concentration 10 μM) was added. The fluorescence intensity kinetics of the product were analyzed using a microplate reader. Protein concentration was determined using a BCA kit. ALDH2 activity in each tissue was represented by the rate of change in fluorescence intensity per unit of protein. The results are shown below. Figure 8 As shown, Figure 8 The results show the enzyme activity of fluorescent probe I-9 in tissue homogenates from 6-month-old mice. Figure 8 It can be seen that probe I-9 can detect the ALDH2 enzyme activity in each homogenate, and the enzyme activity is the highest in mouse liver tissue, which is consistent with the results reported in the literature.

[0161] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A fluorescent probe, characterized in that, It has the structure shown in Equation I: In Equation I, R is R1 can be hydroxyl, amino, substituted amino, cycloalkylamine, or heterocycloalkylamine.

2. The fluorescent probe according to claim 1, characterized in that, The substituted amino group is formed by replacing two hydrogen atoms on the amino group with an alkyl group, wherein the alkyl group is a straight-chain alkyl group having 1 to 6 carbon atoms.

3. The fluorescent probe according to claim 1, characterized in that, The cycloalkylamine group is 4. The fluorescent probe according to claim 1, characterized in that, The heterocyclic alkylamine group is 5. The fluorescent probe according to any one of claims 1 to 4, characterized in that, The specific structure is as follows:

6. The method for preparing the fluorescent probe according to any one of claims 1 to 5, characterized in that, Includes the following steps: The precursor compound shown in Formula 2, ((1,3-dioxacyclopentan-2-yl)methyl)triphenylphosphonium bromide, 18-crown ether-6, and potassium carbonate were subjected to a first reaction to give an intermediate; The intermediate was deprotected to obtain the fluorescent probe; R-CHO Formula 2.

7. The preparation method according to claim 6, characterized in that, The molar ratio of the precursor compound to ((1,3-dioxane-2-yl)methyl)triphenylphosphonium bromide is 1:1 to 1:2, the molar ratio of the precursor compound to 18-crown ether-6 is 1:1 to 1:2, and the molar ratio of the precursor compound to potassium carbonate is 1:1 to 1:2; the first reaction is carried out under nitrogen protection and reflux; the reaction time is 12 to 24 hours.

8. The preparation method according to claim 6, characterized in that, The deprotection reagent is a hydrochloric acid solution with a concentration of 0.5–1.5 mol / L; the deprotection temperature is an ice-water bath for 15–60 min.

9. The use of the fluorescent probe according to any one of claims 1 to 5 in the preparation of reagents for detecting ALDH2 enzyme activity.

10. The application according to claim 9, characterized in that, The reagents for detecting ALDH2 enzyme activity are used in the form of test strips, probe solutions, or kits.

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