Fluorescent probe for identifying D-galactosamine, preparation method and identification method

Abstract

The invention discloses a fluorescent probe for identifying D-galactosamine as well as a preparation method and an identification method thereof, and belongs to the technical field of chemical analysis and detection. The fluorescent probe takes BINOL as a skeleton, and the preparation method of the fluorescent probe comprises the following steps: sequentially synthesizing intermediates (R)-2, (R)-3 and (R)-4, dissolving (R)-4 in a mixed solution of absolute ethyl alcohol and dichloromethane, adding hydrochloric acid, reacting overnight, quenching, separating a water phase and an organic phase, removing a solvent under reduced pressure, and purifying by a silica gel column to obtain a yellow solid product (R)-5, namely the fluorescent probe. The fluorescent probe disclosed by the invention is simple to prepare and low in cost, the D-galactosamine (D-GalN) can be identified from various amino sugars and amino acids in acetonitrile, and the detection method is simple to operate, high in sensitivity and convenient in post-treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical analysis and detection, and particularly relates to a fluorescence probe for identifying D-galactosamine, a preparation method thereof, and an identification method thereof. Background Art

[0002] Amino sugars are a general term for a class of compounds in which the hydroxyl groups of sugars are replaced by amino groups, and are components of many biological macromolecules such as chitosan, antibiotics, lipopolysaccharides, glycoproteins, and mucopolysaccharides. Amino sugars are derived from microorganisms and have high stability. They are components of the cell walls of soil microorganisms. The main amino sugars in soil are: D-galactosamine (D-GalN), D-glucosamine (D-GluN), D-mannosamine (D-ManN), and D-galactosamine is one of the amino sugars rich in the environment. It mainly comes from bacteria and fungi. D-galactosamine is widely used as a microbial marker in the research of soil organic matter accumulation, microbial community dynamics, and carbon and nitrogen cycles. Moreover, D-galactosamine is also an important component of extracellular polymers in soil.

[0003] In addition, D-galactosamine can also be used as a liver toxin, and its pathology is similar to that of clinical viral hepatitis. A large dose of D-galactosamine can cause liver failure, and repeated doses will lead to chronic liver damage. Therefore, it can be used to establish an animal model of liver injury, which is of great significance for studying the dosage of drugs, exploring anti-liver injury foods and drugs, the morphological changes of the damaged liver, and the decay of liver function.

[0004] At present, D-galactosamine has been widely used in the biochemical research of animal hepatitis and has been widely studied as a drug targeting mechanism for liver injury. At the same time, D-galactosamine can also be used as an intermediate for various drugs, such as galactosamine acid. In the prior art, there are various methods for detecting D-galactosamine. Traditional detection methods are generally high-performance liquid chromatography, gas chromatography, infrared spectroscopy, etc. However, these methods have the disadvantages of expensive instrument equipment, cumbersome operation, and low sensitivity. Therefore, there is an urgent need to develop a detection and analysis technology with the characteristics of simplicity, sensitivity, and high efficiency for D-galactosamine. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a fluorescence probe for identifying D-galactosamine, a preparation method thereof, and an identification method thereof, so as to solve the problems of expensive instruments and insufficient sensitivity in the current detection of D-galactosamine.

[0006] To achieve the above purpose, the present invention provides the following technical solutions:

[0007] The present invention provides a fluorescence probe that specifically recognizes D-galactosamine (D-GalN), and the structural formula of the fluorescence probe is shown in Formula 1:

[0008]

[0009] The fluorescent probe uses BINOL as the backbone.

[0010] Furthermore, it also includes a preparation method of a fluorescent probe that specifically recognizes D-galactosamine (D-GalN), comprising the following steps,

[0011] S1. Synthesize intermediate (R)-2

[0012] Disperse (R)-BINOL in anhydrous DCM, add N,N-diisopropylethylamine solution at 0 °C, react at room temperature for 3 hours, cool down to 0 °C and add bromomethyl methyl ether, and continue to react at room temperature for 1 hour; then add ultrapure water to quench the reaction at 0 °C, and separate the aqueous phase and organic phase with ethyl acetate. After removing the solvent, purify by silica gel column to obtain the white solid product (R)-2;

[0013] S2. Synthesize intermediate (R)-3

[0014] Add n-BuLi to the THF solution dissolving (R)-2 at 0 °C, react for 3 hours, add DMF at 0 °C, react at room temperature for 2 hours, then add saturated ammonium chloride solution to quench the reaction, separate the aqueous phase and organic phase, and remove the solvent under reduced pressure. After purification by silica gel column, obtain the yellow solid product (R)-3;

[0015] S3. Synthesize intermediate (R)-4

[0016] Dissolve (R)-3 and anhydrous potassium carbonate in anhydrous acetonitrile, heat to 80 °C and react for 3 h, then add 3-(bromomethyl)phenylboronic acid and reflux overnight; then remove the solvent under reduced pressure, add DCM and H 2 O for extraction, then separate the aqueous phase and organic phase, and remove the solvent under reduced pressure. After purification by silica gel column, obtain the yellow solid product (R)-4;

[0017] S4. Synthesize the fluorescent probe (R)-5

[0018] Dissolve (R)-4 in a mixed solution of anhydrous ethanol and dichloromethane, add hydrochloric acid and react overnight, add solid NaHCO 3 to quench, separate the aqueous phase and organic phase again, remove the solvent under reduced pressure, and after purification by silica gel column, obtain the yellow solid product (R)-5, which is the fluorescent probe.

[0019] Furthermore, in the step S1, the eluent used for column chromatography purification is petroleum ether:ethyl acetate, and their volume ratio is 30:1.

[0020] Furthermore, in the step S2, the eluent used for column chromatography purification is petroleum ether:ethyl acetate, and their volume ratio is 10:1.

[0021] Further, in the step S3, the eluent used in column chromatography purification is petroleum ether: ethyl acetate, and their volume ratio is 1:1.

[0022] Further, in the step S4, the eluent used in column chromatography purification is dichloromethane: methanol, and their volume ratio is 100:1.

[0023] Further, for the method for rapidly and specifically identifying D-galactosamine in acetonitrile using the fluorescent probe, the identification method includes the following steps:

[0024] Dissolve (R)-5 in DMSO to prepare a mother liquor 1 with a concentration of 1.6 mM, dissolve D-galactosamine hydrochloride in a NaHCO 3 solution with the same concentration as the amino sugar hydrochloride to prepare a mother liquor 2 with a concentration of 14.4 mM, and dissolve zinc(II) in ultrapure water to prepare a mother liquor 3 with a concentration of 1.6 mM;

[0025] Sequentially add the mother liquor 1, then acetonitrile, and then sequentially add the mother liquor 2 and the mother liquor 3 into a centrifuge tube, and then immediately mix evenly and react for 2 h;

[0026] After the reaction is completed, add acetonitrile to make up the volume and directly perform fluorescence spectrum detection.

[0027] The beneficial effects of the present invention are as follows:

[0028] 1. The fluorescent probe of the present invention is simple to prepare and has low cost. The raw material BINOL is first treated with bromomethyl methyl ether to obtain the protected intermediate (R)-2, and then deprotonated with an excessive amount of n-butyllithium and formylated with DMF to obtain the intermediate (R)-3. Then, in the presence of anhydrous potassium carbonate, it reacts with 3-(bromomethyl)phenylboronic acid to obtain the intermediate (R)-4. Finally, the protecting group is removed by HCl to form the fluorescent probe (R)-5.

[0029] 2. The aldehyde group on the fluorescent probe (R)-5 of the present invention first reacts with the amino group in the amino sugar to form an imine, and then the phenylborate anion undergoes a substitution reaction with the hydroxyl group of the amino sugar, followed by a ring closure reaction to form a stable phenylborate ester bond, and then coordinates with zinc(II) to produce a strong fluorescence enhancement, thereby identifying D-galactosamine.

[0030] 3. The detection method provided by the present invention is simple to operate, has high sensitivity, and is convenient for post-treatment.

[0031] Other advantages, objectives, and features of the present invention will be described in the subsequent specification, and to some extent, they are obvious to those skilled in the art, or those skilled in the art can be taught from the practice of the present invention. The objectives and other advantages of the present invention can be achieved and obtained through the following specification. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to make the objectives, technical solutions and beneficial effects of the invention clearer, the following drawings are provided for the description of the present invention:

[0033] Figure 1 (a) is the 1H NMR spectrum of (R)-5 of the present invention;

[0034] Figure 1 (b) is the 13C NMR spectrum of (R)-5 of the present invention;

[0035] Figure 2 (a) is the fluorescence spectrum of (R)-5 of the present invention for identifying 3 amino sugars;

[0036] Figure 2 (b) is the fluorescence spectrum of (R)-5 of the present invention for identifying D-galactosamine among 20 amino acids and 3 amino sugars;

[0037] Figure 3 (a) is the fluorescence spectrum of the reaction of (R)-5 of the present invention with D-galactosamine at different times;

[0038] Figure 3 (b) is the fluorescence spectrum of the reaction of (R)-5 of the present invention with D-mannosamine at different times;

[0039] Figure 3 (c) is the fluorescence spectrum of the reaction of (R)-5 of the present invention with D-glucosamine at different times;

[0040] Figure 3 (d) is the fluorescence intensity graph at 550 nm of the reaction of (R)-5 of the present invention with 3 amino sugars at different times;

[0041] Figure 4 (a) is the fluorescence spectrum of (R)-5 of the present invention after reacting with D-galactosamine;

[0042] Figure 4 (b) is the fluorescence spectrum of (R)-5 of the present invention after reacting with D-mannosamine;

[0043] Figure 4 (c) is the fluorescence spectrum of (R)-5 of the present invention after reacting with D-glucosamine;

[0044] Figure 4 (d) is the fluorescence intensity graph at 550 nm of (R)-5 of the present invention after reacting with amino sugars;

[0045] Figure 5 (a) is for (R)-5 of the present invention reacting with 9 equivalents of D-galactosamine in different Zn 2+Fluorescence spectra of the reactions in the system;

[0046] Figure 5 (b) Fluorescence spectra of the reaction of the present invention's (R)-5 with 9 equivalents of D - aminomannose in different Zn 2+ systems;

[0047] Figure 5 (c) Fluorescence spectra of the reaction of the present invention's (R)-5 with 9 equivalents of D - glucosamine in different Zn 2+ systems;

[0048] Figure 5 (d) Fluorescence intensity diagram at 550 nm after the reaction of the present invention's (R)-5 with 9 equivalents of 3 amino sugars in Zn 2+ system. Detailed implementation manners

[0049] As Figures 1 - 5 shown, the present invention provides a fluorescence probe for specifically recognizing D - galactosamine, a preparation method, and a recognition method.

[0050] D - galactosamine is abbreviated as (D - GalN).

[0051] Example 1:

[0052] Synthesize the fluorescence probe (R)-5. All

[0053] S1. Under a nitrogen atmosphere, disperse (R)-BINOL (10.0 g, 34.9 mmol) in anhydrous DCM, cool to 0 °C, add N,N - diisopropylethylamine solution (13 mL, 76.8 mmol), raise the temperature to room temperature and react for 3 hours. Then cool to 0 °C again, add bromomethyl methyl ether (3.13 mL, 38.4 mmol), raise the temperature to room temperature and react for 1 hour. Then cool to 0 °C again and add ultrapure water to quench the reaction. Separate the aqueous phase and organic phase with ethyl acetate. After removing the solvent, purify by silica gel column to obtain the white solid product (R)-2;

[0054] S2. Under a nitrogen atmosphere, add n - BuLi (45.7 mL, 73.2 mmol) to the THF solution containing (R)-2 (6.00 g, 18.3 mmol) at 0 °C. After reacting for 3 hours, cool to 0 °C and add DMF (2.11 mL, 27.4 mmol). Raise the temperature to room temperature and react for 2 hours, then add saturated ammonium chloride solution to quench the reaction. Separate the aqueous phase and organic phase, and remove the solvent under reduced pressure. Purify by silica gel column to obtain the yellow solid product (R)-3;

[0055] S3. Under a nitrogen atmosphere, dissolve (R)-3 (300 mg, 0.84 mmol) and anhydrous potassium carbonate (697 mg, 5 mmol) in anhydrous acetonitrile, heat to 80 °C and react for 3 h. Then add 3-(bromomethyl)phenylboronic acid (270.7 mg, 1.26 mmol) and reflux overnight. Remove the solvent under reduced pressure, add DCM and H 2 O for extraction, separate the aqueous and organic phases, remove the solvent under reduced pressure, and purify by silica gel column chromatography to obtain the yellow solid product (R)-4;

[0056] S4. Dissolve (R)-4 (100 mg, 0.2 mmol) in a mixed solution of anhydrous ethanol and dichloromethane, add 0.5 mL of hydrochloric acid and react overnight. Then add solid NaHCO 3 to quench, separate the aqueous and organic phases, remove the solvent under reduced pressure, and purify by silica gel column chromatography to obtain the yellow solid product (R)-5.

[0057] The nuclear magnetic resonance data of (R)-5 are as follows:

[0058] 1 H NMR (400 MHz, DMSO-d 6 ) δ 10.32 (s, 1H), 10.25 (s, 1H), 8.62 (s, 1H), 8.12 (d, J = 7.2 Hz, 1H), 8.06 (d, J = 9.1 Hz, 1H), 7.96 (d, J = 14.0 Hz, 3H), 7.66–7.58 (m, 3H), 7.45–7.38 (m, 2H), 7.34 (d, J = 6.6 Hz, 1H), 7.27 (s, 1H), 7.10 (t, J = 7.7 Hz, 1H), 7.05–6.96 (m, 3H), 5.15 (s, 2H). 13 C NMR (101 MHz, DMSO-d 6 ) δ 197.46, 154.63, 153.40, 137.30 (d, J = 20.4 Hz), 136.55, 133.72 (d, J = 7.1 Hz), 133.35, 130.59 (d, J = 6.7 Hz), 130.25, 129.41, 129.02, 128.60, 127.66, 127.10, 124.88 (d, J = 13.4 Hz), 124.57, 124.11, 123.15, 118.06 (d, J = 16.6 Hz), 116.26, 70.83.

[0059] In step S1 of the present invention, it was found by thin layer chromatography spotting that the product spot was close to the generated impurity spot. Therefore, the eluent used in column chromatography purification was petroleum ether:ethyl acetate, and their volume ratio was 30:1 to better separate the product spot;

[0060] In step S2, it was found by thin-layer chromatography spotting that the product spot and the impurity spot were farther apart compared to step S1. Therefore, the eluent used for column chromatography purification was petroleum ether:ethyl acetate, with a volume ratio of 10:1, in order to better separate the product spot.

[0061] In step S3, it was found by thin-layer chromatography spotting that the product spot and the impurity spot were even farther apart compared to step S2. Therefore, the eluent used for column chromatography purification was petroleum ether:ethyl acetate, with a volume ratio of 1:1, in order to better separate the product spot.

[0062] Compared to step S3, in step S4, it was found by thin-layer chromatography spotting that the product spot and the impurity spot were farther apart. The eluent used for column chromatography purification was dichloromethane:methanol, with a volume ratio of 100:1, in order to better separate the product spot.

[0063] In each step, the different proportions of the eluent were set in order to better separate the product spot.

[0064] Test:

[0065] 1. Specific selectivity test of (R)-5

[0066] 50 μL of the mother liquor was pipetted from the standard sample of (R)-5 and transferred to a clean 5 mL centrifuge tube. Then, 250 μL of acetonitrile, 50 μL of 14.4 mM (9 equivalents) of amino sugar (3) or amino acid (39) were successively pipetted into the centrifuge tube. At the same time, 50 μL of zinc acetate solution was quickly pipetted into the centrifuge tube.

[0067] After gently shaking and mixing, it was left to stand at room temperature for 2 h. The reaction solution was made up to 4 mL in volume. Immediately after dilution, the solution was transferred to a cuvette for fluorescence spectroscopy test.

[0068] The results are as Figure 2 shown: Figure 2 (a) shows that among the 3 amino sugars for (R)-5, only D-galactosamine produced a very strong fluorescence enhancement at 550 nm, and the fluorescence enhancement of other amino sugars was weak or there was no fluorescence enhancement.

[0069] Figure 2 (b) shows that among the 39 interfering amino acids and 3 amino sugars for (R)-5, only D-galactosamine produced a very strong fluorescence enhancement at 550 nm, which proves that the probe (R)-5 can effectively and specifically recognize D-galactosamine and distinguish D-galactosamine from other amino sugars.

[0070] 2. Reaction time test of (R)-5

[0071] Pipette 50 μL of the mother liquor from the standard sample of (R)-5 and transfer it to a clean 5-mL centrifuge tube. Subsequently, pipette 250 μL of acetonitrile and 50 μL of the aminoglycan test sample at 14.4 mM (9 equivalents) into the centrifuge tube in sequence. Then, quickly pipette 50 μL of zinc acetate solution and add it to the centrifuge tube.

[0072] After gently mixing by shaking, react at room temperature for 1 min, 5 min, 10 min, 30 min, 1 h, 2 h, 3 h, 4 h, and 6 h respectively. After the reaction is completed, make up the volume of the reaction solution to 4 mL. Immediately transfer the solution to a cuvette for fluorescence spectroscopy testing after dilution.

[0073] As Figure 3 shown, the reactions of aminoglycans (D-galactosamine D-GalN, D-mannosamine D-ManN, D-glucosamine D-GluN) at 1 min, 5 min, 10 min, 30 min, 1 h, 2 h, 3 h, 4 h, and 6 h were studied. The results are as Figure 3 (a)-3(c) show that the fluorescence intensities of the three aminoglycans increase with the increase of time. While Figure 3 (d) shows that a plateau is reached after 2 h of reaction and the fluorescence intensity tends to be stable. Thus, the reaction time is determined to be 2 h.

[0074] 3. Equivalent test of aminoglycan of (R)-5

[0075] Pipette 50 μL of the mother liquor from the standard sample of (R)-5 and transfer it to a clean 5-mL centrifuge tube. Then, pipette 250 μL of acetonitrile, and respectively pipette 50 μL of aminoglycan at 0.8 mM, 1.6 mM, 2.4 mM, 3.2 mM, 4.0 mM, 4.8 mM, 6.4 mM, 8.0 mM, 9.6 mM, 11.2 mM, 12.8 mM, 14.4 mM, 16 mM, 17.6 mM, 19.2 mM, 20.8 mM, 22.4 mM, 24 mM, 25.6 mM, 27.2 mM, 28.8 mM, 30.4 mM, 32 mM into the centrifuge tube. At the same time, quickly pipette 50 μL of zinc acetate solution and add it to the centrifuge tube.

[0076] After gently mixing by shaking, let it stand for reaction. Make up the volume of the reaction solution to 4 mL. Immediately transfer the solution to a cuvette for fluorescence spectroscopy testing after dilution.

[0077] As Figure 4The reaction concentrations of three kinds (D-galactosamine D-GalN, D-mannosamine D-ManN, D-glucosamine D-GluN) were studied at 0.8 mM (0.5 eq.), 1.6 mM (1.0 eq.), 2.4 mM (1.5 eq.), 3.2 mM (2.0 eq.), 4.0 mM (2.5 eq.), 4.8 mM (3.0 eq.), 6.4 mM (4.0 eq.), 8.0 mM (5.0 eq.), 9.6 mM (6.0 eq.), 11.2 mM (7.0 eq.), 12.8 mM (8.0 eq.), 14.4 mM (9.0 eq.), 16 mM (10 eq.), 17.6 mM (11 eq.), 19.2 mM (12 eq.), 20.8 mM (13 eq.), 22.4 mM (14 eq.), 24 mM (15 eq.), 25.6 mM (16 eq.), 27.2 mM (17 eq.), 28.8 mM (18 eq.), 30.4 mM (19 eq.), 32 mM (20 eq.). The results are as Figure 4 shown in (a)-4(c), indicating that the fluorescence intensity increases with the increase of the concentration of the three amino sugars. Figure 4 (d) Further shows the peak intensity at λ = 550 nm after the probe (R)-5 reacts with amino sugars at different concentrations. When reaching 9 equivalents, the fluorescence intensity reaches a plateau. Thus, it is determined that the concentration of D-galactosamine should be 9 equivalents.

[0078] 4. Zn 2+ Test on the influence of concentration on fluorescence intensity

[0079] Take 50 μL of the stock solution from the standard sample of (R)-5 and transfer it to a clean 5 mL centrifuge tube. Then, successively take 250 μL of acetonitrile and 50 μL of 14.4 mM (9 equivalents) amino sugar and add them to the centrifuge tube. At the same time, quickly take 50 μL of 0.8 mM, 1.6 mM, 2.4 mM, 3.2 mM, 4.0 mM, 4.8 mM, 5.6 mM, and 6.4 mM zinc acetate solution and add them to the centrifuge tube.

[0080] After gently shaking and mixing, let it stand at room temperature. Dilute the reaction solution to 4 mL. Immediately transfer the solution to a cuvette after dilution for fluorescence spectrum testing.

[0081] As Figure 5 shown, the 0.8 mM, 1.6 mM, 2.4 mM, 3.2 mM, 4.0 mM, 4.8 mM, 5.6 mM, and 6.4 mM Zn 2+ concentrations of the three amino sugars were studied. The results are as Figure 5 shown in (a)-5(c). The fluorescence intensities of the three amino sugars increase with the increase of Zn 2+increases with the increase of concentration. When the Zn 2+ equivalent reaches 0.5 eq., the fluorescence intensity reaches a plateau. Figure 5 (d) shows that high concentrations of Zn 2+ will not affect the fluorescence intensity. To ensure the stability of the fluorescence response, 1 eq. of Zn 2+ is selected for fluorescence detection.

[0082] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A fluorescent probe that specifically recognizes D-galactosamine, characterized in that: The structural formula of the fluorescent probe is shown in Formula 1: The fluorescent probe uses BINOL as a skeleton.

2. The fluorescent probe for specific recognition of D-galactosamine according to claim 1, further comprising a method for preparing the fluorescent probe for specific recognition of D-galactosamine (D-GalN), characterized in that: The following steps are included: S1, Synthetic intermediate (R)-2 (R)-BINOL was dispersed in anhydrous DCM, and N,N-diisopropylethylamine solution was added at 0°C, and the mixture was reacted at room temperature for 3 hours. The mixture was cooled to 0°C and bromomethyl methyl ether was added, and the mixture was heated to room temperature and the reaction was continued for 1 hour. Ultrapure water was then added at 0°C to quench the reaction, and the aqueous phase and the organic phase were separated by ethyl acetate. After the solvent was removed, the mixture was purified by silica gel column to obtain a white solid product (R)-2. S2, Synthetic Intermediate (R)-3 n-BuLi was added to a THF solution containing (R)-2 at 0°C, and DMF was added at 0°C after reacting for 3 hours. After reacting at room temperature for 2 hours, a saturated ammonium chloride solution was added to quench the reaction. The aqueous and organic phases were separated, and the solvent was removed under reduced pressure and purified by silica gel column to obtain a yellow solid product (R)-3; S3, Synthetic intermediate (R)-4 (R)-3 and anhydrous potassium carbonate were dissolved in anhydrous acetonitrile, heated to 80°C and reacted for 3h, then 3-(bromomethyl)phenylboronic acid was added and refluxed to react overnight; then the solvent was removed under reduced pressure, DCM and H2O were added for extraction, and then the aqueous phase and the organic phase were separated, the solvent was removed under reduced pressure, and then purified by silica gel column to obtain a yellow solid product (R)-4; S4, Synthetic fluorescent probe (R)-5 (R)-4 was dissolved in a mixed solution of anhydrous ethanol and dichloromethane, hydrochloric acid was added to react overnight, solid NaHCO3 was added to quench, the aqueous phase and the organic phase were separated again, the solvent was removed under reduced pressure and purified by silica gel column to obtain a yellow solid product (R)-5, which is a fluorescent probe.

3. The method for preparing a fluorescent probe for specific recognition of D-galactosamine according to claim 2, characterized in that: In step S1, the product spot is found to be close to the generated impurity spot by thin layer chromatography, so the eluent used in column chromatography purification is petroleum ether:ethyl acetate, and the volume ratio thereof is 30:1, so as to better separate the product spot.

4. The method for preparing a fluorescent probe for specifically recognizing D-galactosamine according to claim 2, characterized in that: In step S2, the thin layer chromatography plate was used to find that the product point and the impurity point were farther apart than those in step S1, so the eluent used for column chromatography purification was petroleum ether:ethyl acetate, with a volume ratio of 10:1, so as to better separate the product points.

5. The method for preparing a fluorescent probe that specifically recognizes D-galactosamine (D-GalN) according to claim 2, characterized in that: In step S3, the product spots are found to be farther away from the impurity spots by thin layer chromatography than in step S2. The eluent used for column chromatography purification is petroleum ether:ethyl acetate, and the volume ratio thereof is 1:1, so as to better separate the product spots.

6. The method for preparing a fluorescent probe for specific recognition of D-galactosamine according to claim 2, characterized in that: In step S4, the thin layer chromatography plate was used to find that the product spots were farther away from the impurity spots than in step S3. The eluent used for column chromatography purification was dichloromethane:methanol, and the volume ratio was 100:1, so as to better separate the product spots.

7. A method for rapidly and specifically identifying D-galactosamine in acetonitrile using a fluorescent probe according to any one of claims 1 to 6, characterized in that: The identification method comprises the following steps: (R)-5 was dissolved in DMSO to prepare a 1.6 mM mother solution 1, D-galactosamine hydrochloride was dissolved in a NaHCO3 solution of equal concentration to that of amino sugar hydrochloride to prepare a 14.4 mM mother solution 2, and zinc (II) was dissolved in ultrapure water to prepare a 1.6 mM mother solution 3; Add mother solution 1, acetonitrile, mother solution 2, and mother solution 3 to the centrifuge tube in sequence, then mix well and react for 2 hours; After the reaction was completed, acetonitrile was added to make up the volume and fluorescence spectrum detection was performed directly.

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