A fluorescent probe for recognizing d-galactosamine, a preparation method and a recognition method thereof

Abstract

The application discloses a fluorescent probe for identifying D-galactosamine, a preparation method and an identification method, and belongs to the technical field of chemical analysis and detection. The fluorescent probe takes BINOL as a skeleton, and the steps of the fluorescent probe are as follows: sequentially synthesizing intermediates (R)-2, (R)-3 and (R)-4, then dissolving (R)-4 in a mixed solution of anhydrous ethanol and dichloromethane, adding hydrochloric acid and reacting overnight, separating water phase and organic phase after quenching, removing the solvent under reduced pressure, and purifying the yellow solid product (R)-5 through a silica gel column to obtain the fluorescent probe. The fluorescent probe is simple in preparation, low in cost, can identify D-galactosamine (D-GalN) from a plurality of amino sugars and amino acids in acetonitrile, and has the advantages of simple operation, high sensitivity and convenient post-processing.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of chemical analysis and detection, and particularly relates to a fluorescent probe for recognizing D-galactosamine, a preparation method and a recognition method. BACKGROUND

[0002] Amino sugar is a general term for compounds in which the hydroxyl group of a sugar is replaced by an amino group, and is a component of many biological macromolecules such as chitosan, antibiotics, lipopolysaccharides, glycoproteins and mucopolysaccharides. Amino sugar is derived from microorganisms and has high stability, and is a component of soil microbial cell walls. The amino sugars in soil mainly include D-galactosamine (D-GalN), D-glucosamine (D-GluN) and D-mannosamine (D-ManN), and D-galactosamine is one of the amino sugars with high content in the environment, which is mainly derived from bacteria and fungi. D-galactosamine is used as a microbial marker in the study of soil organic matter accumulation, microbial community dynamics and carbon and nitrogen cycles, and D-galactosamine is also an important component of extracellular polymers in soil.

[0003] In addition, D-galactosamine can also be used as a hepatotoxic agent, and its pathology is similar to that of viral hepatitis. A large dose of D-galactosamine can cause liver failure, and repeated doses can cause chronic liver damage, so it can be used to build an animal model of liver damage, which is of great significance for the study of drug dosage, the development of anti-liver damage food and drugs, the morphological changes of damaged liver and the decline of liver function.

[0004] At present, D-galactosamine has been widely used in biochemical research of animal hepatitis, and is widely studied as a drug targeting mechanism for liver damage. At the same time, D-galactosamine can also be used as an intermediate for a variety of drugs, such as amino galactosamine acid. There are many methods for detecting D-galactosamine in the prior art, and traditional detection methods generally include high performance liquid chromatography, gas chromatography and infrared spectroscopy. However, these methods have the disadvantages of expensive instruments and equipment, complicated operation and low sensitivity. Therefore, it is urgent to develop a detection and analysis technology for D-galactosamine with the characteristics of simplicity, sensitivity and high efficiency. SUMMARY

[0005] Therefore, the purpose of the present application is to provide a fluorescent probe for recognizing D-galactosamine, a preparation method and a recognition method, so as to solve the problem of expensive instruments and insufficient sensitivity in the detection of D-galactosamine.

[0006] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0007] The present application provides a fluorescent probe for specifically recognizing D-galactosamine (D-GalN), and the structural formula of the fluorescent probe is shown as formula 1:

[0008]

[0009] The fluorescent probe takes BINOL as a skeleton.

[0010] Further, the application further provides a preparation method of the fluorescent probe for specifically recognizing D-galactosamine (D-GalN), comprising the following steps,

[0011] S1, synthesis of intermediate (R)-2

[0012] (R)-BINOL is dispersed in anhydrous DCM, N,N-diisopropyl ethylamine solution is added at 0°C, and reaction is carried out at room temperature for 3 hours, and then the reaction is quenched by adding ultrapure water at 0°C, and the organic phase is separated from the aqueous phase by using ethyl acetate, and the white solid product (R)-2 is obtained after removing the solvent and purifying through a silica gel column.

[0013] S2, synthesis of intermediate (R)-3

[0014] n-BuLi is added to the THF solution in which (R)-2 is dissolved at 0°C, and after reaction for 3 hours, DMF is added at 0°C, and after reaction for 2 hours at room temperature, the reaction is quenched by adding saturated ammonium chloride solution, the organic phase is separated from the aqueous phase, and the yellow solid product (R)-3 is obtained after removing the solvent under reduced pressure and purifying through a silica gel column.

[0015] S3, synthesis of intermediate (R)-4

[0016] (R)-3 is dissolved in anhydrous acetonitrile with anhydrous potassium carbonate, heated to 80°C and reacted for 3 hours, and then 3-(bromomethyl)phenylboronic acid is added to reflux overnight; then the solvent is removed under reduced pressure, DCM and H2O are added for extraction, and then the organic phase is separated from the aqueous phase, and the yellow solid product (R)-4 is obtained after removing the solvent under reduced pressure and purifying through a silica gel column.

[0017] S4, synthesis of fluorescent probe (R)-5

[0018] (R)-4 is dissolved in a mixed solution of anhydrous ethanol and dichloromethane, hydrochloric acid is added for reaction overnight, solid NaHCO3 is added for quenching, the organic phase is separated from the aqueous phase again, the yellow solid product (R)-5 is obtained after removing the solvent under reduced pressure and purifying through a silica gel column, and the product is the fluorescent probe.

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

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

[0021] Further, in the step S3, the eluent used in the column chromatography is petroleum ether: ethyl acetate with a volume ratio of 1:1.

[0022] Further, in the step S4, the eluent used in the column chromatography is dichloromethane: methanol with a volume ratio of 100:1.

[0023] Further, the method for rapidly and specifically recognizing D-galactosamine by the fluorescent probe in acetonitrile comprises the following steps:

[0024] Dissolve (R)-5 in DMSO to obtain a mother liquor 1 with a concentration of 1.6 mM, dissolve D-galactosamine hydrochloride in a NaHCO3 solution with the same concentration of the amino sugar hydrochloride to obtain a mother liquor 2 with a concentration of 14.4 mM, and dissolve zinc (II) in ultrapure water to obtain a mother liquor 3 with a concentration of 1.6 mM;

[0025] Add the mother liquor 1, then acetonitrile, and then the mother liquor 2 and the mother liquor 3 in sequence in a centrifugal tube, and then immediately mix and react for 2 h;

[0026] After the reaction is completed, acetonitrile is added to constant volume, and then fluorescence spectrum detection is directly performed.

[0027] The present application has the following beneficial effects:

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

[0029] 2. The aldehyde group on the fluorescent probe (R)-5 is first reacted with the amino group in the amino sugar to generate an imine, then the phenylboronic acid anion is substituted with the hydroxyl group in the amino sugar to generate a stable phenylboronic acid ester bond, and then the zinc (II) is coordinated to generate a strong fluorescence enhancement, so that the D-galactosamine is recognized.

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

[0031] Other advantages, objects, and features of the present application will be set forth in the following specification, and in part will become apparent to those skilled in the art from the present application, or can be learned by practice of the present application. The objects and other advantages of the present application can be realized and obtained by the following description. BRIEF DESCRIPTION OF DRAWINGS

[0032] In order to make the purpose, technical scheme and beneficial effects of the application more clear, the application provides the following figures for illustration.

[0033] Figure 1 (a) is a nuclear magnetic resonance hydrogen spectrum of (R)-5 of the application;

[0034] Figure 1 (b) is a nuclear magnetic resonance carbon spectrum of (R)-5 of the application;

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

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

[0037] Figure 3 (a) is a fluorescence spectrum of (R)-5 of the application reacting with D-galactosamine at different time;

[0038] Figure 3 (b) is a fluorescence spectrum of (R)-5 of the application reacting with D-mannosamine at different time;

[0039] Figure 3 (c) is a fluorescence spectrum of (R)-5 of the application reacting with D-glucosamine at different time;

[0040] Figure 3 (d) is a fluorescence intensity graph of (R)-5 of the application reacting with 3 amino sugars at 550 nm at different time;

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

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

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

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

[0045] Figure 5 (a) is a fluorescence spectrum of (R)-5 of the application reacting with 9 equivalents of D-galactosamine in different Zn 2+ systems;

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

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

[0048] Figure 5 (d) Fluorescence intensity at 550 nm after the reaction of (R)-5 with 9 equivalents of 3 amino sugars in Zn 2+ systems. DETAILED DESCRIPTION

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

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

[0051] Example 1:

[0052] Synthesis of fluorescent probe (R)-5, all

[0053] S1, under nitrogen atmosphere, (R)-BINOL (10.0 g, 34.9 mmol) was dispersed in anhydrous DCM, cooled to 0°C, and N,N-diisopropyl ethylamine solution (13 mL, 76.8 mmol) was added, and the reaction was carried out at room temperature for 3 hours, then cooled to 0°C again, and bromomethyl methyl ether (3.13 mL, 38.4 mmol) was added, and the reaction was carried out at room temperature for 1 hour; then cooled to 0°C again, and quenched with ultrapure water, separated the organic phase from the aqueous phase with ethyl acetate, and after removing the solvent, the white solid product (R)-2 was obtained by silica gel column purification;

[0054] S2, under nitrogen atmosphere, n-BuLi (45.7 mL, 73.2 mmol) was added to a THF solution containing (R)-2 (6.00 g, 18.3 mmol) at 0°C, and the reaction was carried out for 3 hours, then cooled to 0°C, and DMF (2.11 mL, 27.4 mmol) was added, and the reaction was carried out at room temperature for 2 hours, then quenched with saturated ammonium chloride solution, separated the organic phase from the aqueous phase, and after removing the solvent under reduced pressure, the yellow solid product (R)-3 was obtained by silica gel column purification;

[0055] S3, (R)-3 (300 mg, 0.84 mmol) was dissolved in anhydrous acetonitrile with anhydrous potassium carbonate (697 mg, 5 mmol) under nitrogen atmosphere, heated to 80°C for 3h, then 3-(bromomethyl)benzeneboronic acid (270.7 mg, 1.26 mmol) was added and refluxed overnight; the solvent was removed under reduced pressure, DCM and H2O were added for extraction, the aqueous phase was separated from the organic phase, the solvent was removed under reduced pressure, and the yellow solid product (R)-4 was obtained after silica gel column purification;

[0056] S4, (R)-4 (100 mg, 0.2 mmol) was dissolved in a mixed solution of anhydrous ethanol and dichloromethane, 0.5 mL of hydrochloric acid was added and reacted overnight, then solid NaHCO3 was added to quench and separate the aqueous phase from the organic phase, the solvent was removed under reduced pressure, and the yellow solid product (R)-5 was obtained after silica gel column purification.

[0057] The nuclear magnetic resonance data of (R)-5 is:

[0058] 1 H NMR (400 MHz, DMSO-d6) 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-d6) 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 application, thin layer chromatography is used to find that the product point is close to the generated impurity point, so the eluent used in column chromatography purification is petroleum ether: ethyl acetate, the volume ratio is 30:1, so as to better separate the product point;

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

[0061] In step S3, the product spot and impurity spot were found to be farther apart than in step S2 using thin-layer chromatography. Therefore, the eluent used in column chromatography purification was petroleum ether: ethyl acetate in a volume ratio of 1:1, in order to better separate the product spot.

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

[0063] The different eluent ratios in each step are all for the purpose of better separating the product spots.

[0064] test:

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

[0066] Take 50 μL of the stock solution from the (R)-5 standard sample and transfer it to a clean 5 mL centrifuge tube. Then, take 250 μL of acetonitrile and 50 μL of 14.4 mM (9 equivalents) amino sugar (3 pieces) or amino acid (39 pieces) and add them to the centrifuge tube in sequence. At the same time, quickly take 50 μL of zinc acetate solution and add it to the centrifuge tube.

[0067] After gentle shaking and mixing, let stand at room temperature for 2 hours, then dilute the reaction solution to 4 mL. Immediately after dilution, transfer the solution to a cuvette for fluorescence spectroscopy testing.

[0068] The results are as follows Figure 2 As shown: Figure 2 (a) shows that among the three amino sugars, only D-galactosamine of (R)-5 produces a very strong fluorescence enhancement at 550 nm, while the other amino sugars show weak or no fluorescence enhancement.

[0069] Figure 2 (b) shows that among the 39 interfering amino acids and 3 amino sugars, only D-galactosamine of (R)-5 exhibits very strong fluorescence enhancement at 550 nm, which proves that 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] Take 50 μL of the mother liquor from the standard sample of (R)-5 and transfer it to a clean 5 mL centrifuge tube, then take 250 μL of acetonitrile, 50 μL of 14.4 mM (9 equivalents) of the amino sugar to be tested, and 50 μL of zinc acetate solution in sequence and add them to the centrifuge tube;

[0072] After mixing by slight shaking, react for 1 min, 5 min, 10 min, 30 min, 1 h, 2 h, 3 h, 4 h, and 6 h at room temperature, respectively, and then dilute the reaction solution to 4 mL. Immediately after dilution, transfer the solution to a cuvette for fluorescence spectrum testing;

[0073] As shown in Figure 3 , the reaction of the amino sugar (D-amino galactose D-GalN, D-amino mannose D-ManN, D-amino glucose D-GluN) was studied for 1 min, 5 min, 10 min, 30 min, 1 h, 2 h, 3 h, 4 h, and 6 h, and the results are shown in Figure 3 (a)-3(c) show that the fluorescence intensity of the three amino sugars increases with time; and Figure 3 (d) shows that a plateau is reached after 2 h of reaction, and the fluorescence intensity tends to be stable, thus determining the reaction time to be 2 h.

[0074] 3. Amino sugar equivalent test of (R)-5

[0075] Take 50 μL of the mother liquor from the standard sample of (R)-5 and transfer it to a clean 5 mL centrifuge tube, then take 250 μL of acetonitrile, 50 μL of 14.4 mM (9 equivalents) of the amino sugar to be tested, and 50 μL of zinc acetate solution in sequence and add them to the centrifuge tube;

[0076] After mixing by slight shaking, react for 1 min, 5 min, 10 min, 30 min, 1 h, 2 h, 3 h, 4 h, and 6 h at room temperature, respectively, and then dilute the reaction solution to 4 mL. Immediately after dilution, transfer the solution to a cuvette for fluorescence spectrum testing;

[0077] As shown in Figure 4The study of the concentration of 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.) of three (D-galactosamine D-GalN, D-mannosamine D-ManN, D-glucosamine D-GluN) is shown in Figure 4 (a) -5 (c) shows that the fluorescence intensity increases with the increase of the concentration of three amino sugar, Figure 4 (d) further shows that the peak intensity at λ = 550 nm of probe (R)-5 after reacting with different concentrations of amino sugar, when reaching 9 equivalents, the fluorescence intensity reaches a plateau, thus determining that the concentration of D-galactosamine should be 9 equivalents.

[0078] 4, Zn 2+ Effect of concentration on fluorescence intensity test

[0079] 50 μL of the mother liquor was taken from the standard sample of (R)-5 and transferred to a clean 5 mL centrifuge tube, and then 250 μL of acetonitrile and 50 μL of 14.4 mM (9 equivalents) amino sugar were added to the centrifuge tube in turn; at the same time, 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 were quickly taken and added to the centrifuge tube;

[0080] After mixing by slight shaking, the reaction solution was diluted to 4 mL at room temperature, and then the solution was immediately transferred to a cuvette for fluorescence spectrum test;

[0081] As shown in Figure 5 The study 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 Zn 2+ concentration of three amino sugars, the results are shown in Figure 5 (a) -5 (c) shows that the fluorescence intensity of three amino sugars increases with the increase of the concentration of Zn 2+The fluorescence intensity is enhanced with the increase of the concentration of Zn 2+ The fluorescence intensity reached a plateau after the equivalent reached 0.5 eq. Figure 5 (d) It is indicated that high concentration of Zn 2+ will not affect the fluorescence intensity, in order to ensure the stability of the fluorescence response, 1 eq. Zn 2+ participates in the fluorescence detection.

[0082] Finally, it should be pointed out that the above preferred embodiments are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application 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 application.

Claims

1. A method for preparing a fluorescent probe which specifically recognizes D-galactosamine, characterized by: The structural formula of the fluorescent probe is shown as formula 1: Formula 1 The fluorescent probe takes BINOL as a skeleton; The method comprises the following steps, S1, synthesis of intermediate (R)-2 (R)-BINOL is dispersed in anhydrous DCM, N,N-diisopropylethylamine solution is added at 0 DEG C, reaction is carried out at room temperature for 3 hours, cooling is carried out to 0 DEG C, bromomethyl methyl ether is added, reaction is continued at room temperature for 1 hour, then water is added at 0 DEG C to quench the reaction, the organic phase is separated from the aqueous phase by using ethyl acetate, after removing the solvent, a white solid product (R)-2 is obtained by silica gel column purification, the (R)-2 is 2'-(methoxymethoxy)-[1,1'-binaphthalen]-2-ol; S2, synthesis of intermediate (R)-3 n-BuLi is added to the THF solution containing (R)-2 at 0 DEG C, after reaction for 3 hours, DMF is added at 0 DEG C, after reaction for 2 hours at room temperature, saturated ammonium chloride solution is added to quench the reaction, the organic phase is separated from the aqueous phase, after removing the solvent under reduced pressure, a yellow solid product (R)-3 is obtained by silica gel column purification, the (R)-3 is 2'-hydroxy-2-(methoxymethoxy)-[1,1'-binaphthalen]-3-formaldehyde; S3, synthesis of intermediate (R)-4 (R)-3 is dissolved in anhydrous acetonitrile with anhydrous potassium carbonate, heating is carried out to 80 DEG C and reaction is carried out for 3 hours, 3-(bromomethyl)benzene boronic acid is added to carry out reflux reaction overnight, then the solvent is removed under reduced pressure, DCM and H2O are added to carry out extraction, then the organic phase is separated from the aqueous phase, after removing the solvent under reduced pressure, a yellow solid product (R)-4 is obtained by silica gel column purification, the (R)-4 is (3-(((3'-formyl-2'-(methoxymethoxy)-[1,1'-binaphthalen]-2-yl)oxy)methyl)phenyl)boronic acid; S4, synthesis of fluorescent probe (R)-5 (R)-4 is dissolved in a mixed solution of anhydrous ethanol and dichloromethane, hydrochloric acid is added to carry out reaction overnight, solid NaHCO3 is added to quench, the organic phase is separated from the aqueous phase again, after removing the solvent under reduced pressure, a yellow solid product (R)-5 is obtained by silica gel column purification, the (R)-5 is (3-(((3'-formyl-2'-hydroxy-[1,1'-binaphthalenyl]-2-yl)oxy)methyl)phenyl)boronic acid, which is the fluorescent probe. 2.The method for preparing a fluorescent probe for specifically recognizing D-galactosamine according to claim 1, characterized in that: In the step S1, thin layer chromatography is used to find that the product point is close to the impurity point, so petroleum ether: ethyl acetate with a volume ratio of 30:1 is used as the eluent in silica gel column purification, so that the product point can be better separated. 3.The method for preparing a fluorescent probe specifically recognizing D-galactosamine according to claim 2, characterized in that: In the step S2, thin layer chromatography is used to find that the product point is far from the impurity point compared with the step S1, so petroleum ether: ethyl acetate with a volume ratio of 10:1 is used as the eluent in silica gel column purification, so that the product point can be better separated. 4.The method for preparing a fluorescent probe for specifically recognizing D-galactosamine according to claim 3, characterized in that: In the step S3, thin layer chromatography is used to find that the product point is far from the impurity point compared with the step S2, so petroleum ether: ethyl acetate with a volume ratio of 1:1 is used as the eluent in silica gel column purification, so that the product point can be better separated. 5.The method for preparing a fluorescent probe for specifically recognizing D-galactosamine according to claim 4, characterized in that: In step S4, the product spot and the impurity spot are found to be further apart from each other than in step S3 by thin layer chromatography. The eluent used in the silica gel column purification is dichloromethane:methanol in a volume ratio of 100:1, so as to better separate the product spot from the impurity spot.

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