High-sensitivity rapid detection and analysis method for amino sugar

Abstract

The invention discloses a high-sensitivity rapid detection and analysis method of amino sugar, which realizes high-selectivity and high-sensitivity fluorescent recognition of D-galactosamine (D-GalN) by designing and synthesizing a novel chiral fluorescent probe (R)-L10 based on a binaphthol skeleton. The probe is short in synthesis route, simple and convenient to operate and high in yield. Under exciting light of 430 nm, the fluorescent probe reacts with D-GalN to generate a specific fluorescence enhancement signal at 550 nm, and the fluorescent probe is not interfered by amino sugar, chiral amino acid or chiral carbohydrate with similar structures. The method can realize quantitative analysis of D-GalN in the soil extract and real-time fluorescence imaging in cells, has the advantages of good stability, rapid response and strong specificity, and provides an effective tool for detection of D-galactosamine in a complex environment. The fluorescent probe (R)-L10 disclosed by the invention realizes specific fluorescent recognition of 0-30 equivalent of D-galactosamine, is high in detection stability, and can realize determination of the content of D-galactosamine in soil and cells.

Description

Technical Field

[0001] This invention relates to the field of chiral fluorescence detection technology, and to a highly sensitive and rapid detection and analysis method for amino sugars, particularly a method for the specific recognition of D-galactosamine based on the chiral fluorescent probe (R)-L10. Background Technology

[0002] Amino sugars are derivatives formed by replacing the hydroxyl groups with amino groups in sugar molecules. They are widely found in biomolecules such as hyaluronic acid, chondroitin sulfate, and peptidoglycan. D-galactosamine (D-GalN), as a marker of soil microbial cell wall residues, can reflect the structure of soil microbial communities and the source of organic matter. Furthermore, it is associated with diseases such as liver damage and tumors in vivo. Therefore, the high sensitivity and selectivity of D-GalN detection has significant application value.

[0003] While commonly used detection methods such as gas chromatography and high-performance liquid chromatography are mature, they are complex to operate, require expensive equipment, and are difficult to implement in real-time, in-situ detection. Fluorescence detection methods have advantages such as high sensitivity, simple operation, and real-time imaging, but due to the similar structures of D-galactosamine, D-glucosamine, and D-mannose, which differ only in spatial configuration, the specific identification of D-GalN is extremely challenging.

[0004] Binaphenol (BINOL) is widely used in the construction of chiral fluorescent probes due to its stable chiral structure and ease of modification. This invention designs and synthesizes a novel chiral fluorescent probe (R)-L10 based on the BINOL backbone, achieving for the first time highly selective fluorescent recognition of D-GalN, and successfully applying it to the detection of D-GalN in soil and cells. Summary of the Invention

[0005] The purpose of this invention is to provide a highly sensitive and rapid detection and analysis method for amino sugars, which aims to provide a highly selective and sensitive detection method based on the chiral fluorescent probe (R)-L10, so as to achieve rapid and specific quantitative analysis and fluorescence imaging of D-galactosamine in complex environments (such as soil and cells).

[0006] According to the purpose of this invention, a highly sensitive and rapid detection and analysis method for amino sugars is provided, comprising the following steps: S1, Synthetic fluorescent probe (R)-L10: S101. Weigh (R)-binaphthol and dissolve it in ultra-dry dichloromethane. Under 0°C conditions, slowly add N,N-diisopropylethylamine, then slowly add bromomethyl methyl ether. After the reaction is complete, add ultrapure water to quench the reaction, extract with dichloromethane, combine the organic phases, dry with anhydrous sodium sulfate, purify by column chromatography, and vacuum dry to obtain white intermediate (R)-6. S102. Weigh (R)-6 and dissolve it in ultra-dry tetrahydrofuran. Cool the solution to -78 °C and add n-butyllithium dropwise for 2 h. Then add ultra-dry N-N dimethylformamide dropwise. After the reaction is complete, add saturated ammonium chloride solution to quench the reaction under ice-water bath conditions. Extract with ethyl acetate, combine the organic phases, dry with anhydrous sodium sulfate, and purify by column chromatography to obtain intermediate (R)-7. S103. Weigh intermediate (R)-7 into a two-necked flask, evacuate and purge with nitrogen three times, add anhydrous acetonitrile to fully dissolve the raw material, then add naphthalimide fragment (8) and anhydrous K2CO3, heat under reflux at 80℃, after the reaction is complete, restore to room temperature, extract with dichloromethane, combine the organic phases, dry with anhydrous sodium sulfate, and purify by column chromatography to obtain yellow intermediate (R)-L9. S104. Weigh (R)-9, add dichloromethane and concentrated hydrochloric acid, stir thoroughly until the reaction is complete, then add sodium bicarbonate to adjust the pH to neutral, extract with dichloromethane, combine the organic phases, dry with anhydrous sodium sulfate, remove the solvent and obtain the fluorescent probe (R)-L10. S2. React the fluorescent probe (R)-L10 with the sample to be tested, and achieve specific recognition of D-galactosamine by detecting changes in fluorescence signal; The probe reacts with the sample to be tested, and the qualitative and quantitative analysis of D-GalN is achieved by detecting the change in fluorescence intensity at 550 nm. Specifically, the fluorescence detection in this invention is performed at 550 nm, and the excitation wavelength is 430 nm.

[0007] Further, in step S101, the equivalent ratio of (R)-binaphthol, N,N-diisopropylethylamine and bromomethyl methyl ether is 1:2:1.2.

[0008] Furthermore, in step S101, the eluent used during silica gel column purification consists of petroleum ether and ethyl acetate in a volume ratio of 10:1.

[0009] Further, in step S102, the amount of n-butyllithium is 1.5 equivalents, and the amount of N,N dimethylformamide is 1.5 equivalents; the eluent used in silica gel column purification consists of petroleum ether and ethyl acetate in a volume ratio of 5:1.

[0010] Further, in step S103, the amount of anhydrous K2CO3 used is 2 equivalents; the amount of naphthalimide fragment (8) used is 1.5 equivalents; the eluent used in silica gel column purification is composed of petroleum ether and ethyl acetate in a volume ratio of 3:1.

[0011] Furthermore, in step S104, the concentrated hydrochloric acid is 10 equivalents; the eluent used in silica gel column purification consists of petroleum ether and ethyl acetate in a volume ratio of 1:1.

[0012] The beneficial effects of this invention are: This invention provides a chiral fluorescent probe (R)-L10 with a short synthetic route, simple operation, and high yield. This probe has high selectivity and high sensitivity for D-galactosamine (D-GalN), is not affected by other amino sugars, chiral amino acids, or chiral sugars, and can be applied to real-time fluorescence imaging detection of D-GalN in soil extracts and cellular environments, realizing specific, rapid, and stable qualitative and quantitative analysis of D-GalN. Attached Figure Description

[0013] Figure 1 The molecular structural formula of the fluorescent probe in the embodiments of the present invention is shown below; Figure 2 The proton NMR spectrum of the fluorescent probe (R)-L10 in this embodiment of the invention; Figure 3 The carbon NMR spectrum of the fluorescent probe (R)-L10 in this embodiment of the invention; Figure 4 This shows the fluorescence enhancement at 550 nm after the fluorescent probe (R)-L10 of this invention interacts with different chiral analytes; Figure 5 This invention illustrates the effect of reaction time on the fluorescence recognition of D-GalN by probe (R)-L10 in an embodiment of the invention. Figure 6 This illustrates the change in fluorescence enhancement at 550 nm over time when probe (R)-L10 of this invention recognizes three D-amino sugars. Figure 7 This is a graph showing the fluorescence enhancement results of the fluorescent probe (R)-L10 in this embodiment of the invention after recognizing D-GalN solutions of different concentrations; Figure 8 This invention illustrates the fluorescence enhancement at 550 nm as a function of D-amino sugar concentration when probe (R)-L10 fluorescently identifies three D-amino sugars in an embodiment of the present invention. Figure 9 The image shows the fluorescence enhancement results of the fluorescent probe (R)-L10 in this embodiment of the invention in identifying three D-amino sugars in different soils. Figure 10 This is a diagram showing the results of the fluorescent probe (R)-L10 in an embodiment of the present invention recognizing equal amounts of amino sugars with different compositions (the samples consist of D-GalN and D-GluN). Figure 11 This is a diagram showing the results of the fluorescent probe (R)-L10 in an embodiment of the present invention recognizing equal amounts of amino sugars with different compositions (the samples consist of D-GalN and D-ManN). Figure 12 This is a fluorescence imaging result of the fluorescent probe (R)-L10 recognizing three D-amino sugars in cells according to an embodiment of the present invention. Detailed Implementation

[0014] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0015] Example 1 like Figures 1-12 As shown, A highly sensitive and rapid detection and analysis method for amino sugars includes the following steps: S1, Synthetic fluorescent probe (R)-L10: S101. Weigh (R)-binaphthol and dissolve it in ultra-dry dichloromethane. Under 0°C conditions, slowly add N,N-diisopropylethylamine, then slowly add bromomethyl methyl ether. After the reaction is complete, add ultrapure water to quench the reaction, extract with dichloromethane, combine the organic phases, dry with anhydrous sodium sulfate, purify by column chromatography, and vacuum dry to obtain white intermediate (R)-6. S102. Weigh (R)-6 and dissolve it in ultra-dry tetrahydrofuran. Cool the solution to -78 °C and add n-butyllithium dropwise for 2 h. Then add ultra-dry N-N dimethylformamide dropwise. After the reaction is complete, add saturated ammonium chloride solution to quench the reaction under ice-water bath conditions. Extract with ethyl acetate, combine the organic phases, dry with anhydrous sodium sulfate, and purify by column chromatography to obtain intermediate (R)-7. S103. Weigh intermediate (R)-7 into a two-necked flask, evacuate and purge with nitrogen three times, add anhydrous acetonitrile to fully dissolve the raw material, then add naphthalimide fragment (8) and anhydrous K2CO3, heat under reflux at 80℃, after the reaction is complete, restore to room temperature, extract with dichloromethane, combine the organic phases, dry with anhydrous sodium sulfate, and purify by column chromatography to obtain yellow intermediate (R)-L9. S104. Weigh (R)-9, add dichloromethane and concentrated hydrochloric acid, stir thoroughly until the reaction is complete, then add sodium bicarbonate to adjust the pH to neutral. After extraction with dichloromethane, combine the organic phases, dry with anhydrous sodium sulfate, and remove the solvent to obtain the fluorescent probe (R)-L10.

[0016] S2. React the fluorescent probe (R)-L10 with the sample to be tested, and achieve specific recognition of D-galactosamine by detecting changes in fluorescence signal; The probe reacts with the sample to be tested, and the qualitative and quantitative analysis of D-GalN is achieved by detecting the change in fluorescence intensity at 550 nm. Specifically, the fluorescence detection in this invention is performed at 550 nm, and the excitation wavelength is 430 nm.

[0017] Specifically, in step S101, the equivalent ratio of (R)-binaphthol, N,N-diisopropylethylamine and bromomethyl methyl ether is 1:2:1.2.

[0018] In step S101, the eluent used during silica gel column purification consists of petroleum ether and ethyl acetate in a volume ratio of 10:1.

[0019] In step S102, the amount of n-butyllithium is 1.5 equivalents and the amount of N,N dimethylformamide is 1.5 equivalents; the eluent used in silica gel column purification consists of petroleum ether and ethyl acetate in a volume ratio of 5:1.

[0020] In step S103, the amount of anhydrous K2CO3 used is 2 equivalents; the amount of naphthalimide fragment (8) used is 1.5 equivalents; the eluent used in silica gel column purification is composed of petroleum ether and ethyl acetate in a volume ratio of 3:1.

[0021] In step S104, the concentrated hydrochloric acid is 10 equivalents; the eluent used in silica gel column purification consists of petroleum ether and ethyl acetate in a volume ratio of 1:1.

[0022] This embodiment also provides a fluorescent probe (R)-L10 for detecting D-galactosamine, which is synthesized by the method described above. The application of this fluorescent probe (R)-L10 in the preparation of a kit for detecting D-galactosamine.

[0023] This invention provides a method applicable to the detection of D-galactosamine in soil extracts or cellular environments. The probe (R)-L10 of this invention features a short synthetic route, simple operation, and high yield; it exhibits high selectivity and sensitivity for D-GalN, with a detection limit as low as 0.02 mM; it is unaffected by interference from other amino sugars, amino acids, or sugars; and it can be applied to real-time fluorescence imaging detection of D-GalN in soil extracts and cellular environments.

[0024] Example 2 This embodiment provides a method for synthesizing the fluorescent probe (R)-L10, including the following steps: S101: (R)-binaphthol (1 eq) was dissolved in ultra-dry dichloromethane, and N,N-diisopropylethylamine (2 eq) and bromomethyl methyl ether (1.2 eq) were slowly added at 0 °C. After the reaction was completed, the mixture was quenched, extracted, dried, and subjected to column chromatography (petroleum ether: ethyl acetate = 10:1) to obtain a white solid (R)-6.

[0025] S102: Dissolve (R)-6 in ultra-dry tetrahydrofuran, add n-butyllithium (1.5 eq) dropwise at -78℃ for 2 h, then add N,N-dimethylformamide (1.5 eq), quench, extract, dry, and column chromatography (petroleum ether: ethyl acetate = 5:1) to obtain intermediate (R)-7.

[0026] S103: (R)-7 was reacted with naphthalimide fragment (8) (1.5 eq) and anhydrous K2CO3 (2 eq) in anhydrous acetonitrile under reflux at 80 °C, and the reaction was followed up to obtain a yellow solid (R)-L9.

[0027] S104: (R)-L9 was reacted with concentrated hydrochloric acid (10 eq) in dichloromethane, the pH was adjusted to neutral, extracted, dried, and subjected to column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain the fluorescent probe (R)-L10.

[0028] Example 3 This invention provides a fluorescence detection method for D-GalN, comprising the following steps: S1. Dissolve the fluorescent probe in methanol to obtain a 1.6 mM first solution; S2. Dissolve the amino sugar in ultrapure water and add 1 equivalent of NaHCO3 to obtain a second solution of 320 mM. S3. Take 50 μL of each of the first and second solutions and add them to another sample vial; S4. Add 300 μL of methanol to the sample vial, mix well and react for 3 h. S5. After the reaction is complete, dilute to 4 mL with isopropanol to prepare a 0.02 mM third solution. S6. Place the third solution as a sample in a fluorescence spectrometer and use a light source with a fixed excitation wavelength to detect the sample, thereby specifically and selectively identifying D-GalN.

[0029] The fluorescent probe (R)-L10 was reacted with D-galactosamine (D-GalN), D-glucosamine (D-GluN), D-mannose (D-ManN), L / D-serine (Ser), L / D-cysteine ​​(Cys), L / D-proline (Pro), L / D-aspartic acid (Asp), L / D-histidine (His), L / D-glucose (Glu), L / D-galactose (Gal), and L / D-mannose (Man), and the results were as follows: Figure 4 As shown, the fluorescent probe (R)-L10 exhibited specific and selective fluorescence enhancement for D-GalN at an excitation wavelength of 430 nm (emission wavelength of 550 nm), while other analytes showed little or no change.

[0030] Example 3 This embodiment demonstrates how different reaction times affect the recognition performance of the fluorescent probe. To illustrate the effect of reaction time on the recognition of D-GalN by the fluorescent probe (R)-L10, 10 experimental groups were set up with reaction times of 10 minutes, 30 minutes, 1 hour, 1.5 hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, and 7 hours.

[0031] like Figures 5-6 As shown, from Figure 5 It can be seen that during the D-Cys recognition process, the fluorescence intensity gradually increases with the extension of reaction time, but after 3 hours of reaction, the fluorescence enhancement tends to stabilize and is no longer affected by the reaction time. from Figure 6 It can be seen that the fluorescence enhancement trend of (R)-L10 after reacting with D-GluN is consistent with that of D-GalN, and it remains stable within a low fluorescence emission intensity range after 3 hours of reaction. However, the fluorescence enhancement of (R)-L10 after reacting with D-ManN is almost negligible until 7 hours.

[0032] Example 4 The fluorescence enhancement signal of probe (R)-L10 differs when recognizing different concentrations of D-GalN. To demonstrate the effect of analyte concentration on the recognition of D-GalN by fluorescent probe (R)-L10, 12 experimental groups were set up with concentrations of 1 eq., 2 eq., 3 eq., 4 eq., 5 eq., 6 eq., 7 eq., 10 eq., 15 eq., 20 eq., 25 eq., and 30 eq. It was found that even when D-GalN was as low as 0.02 mM (1 eq), there was already a recognition effect. When the D-GalN content increased to 10 eq., the fluorescence enhancement reached its peak, and the intensity no longer increased with the increase of D-GalN concentration.

[0033] from Figure 7 It can be seen that during the recognition process of D-GalN, the fluorescence intensity gradually increases with the increase of D-GalN concentration, and the fluorescence enhancement tends to stabilize when the D-GalN content reaches 10 eq. from Figure 8 It can be seen that when the D-GalN content is less than 10 eq, the fluorescence enhancement at 550 nm shows a linear increasing trend with the D-GalN concentration. When the D-GalN content is greater than 10 eq, the fluorescence enhancement tends to stabilize, achieving high-concentration detection of D-Cys. However, the fluorescence enhancement for D-GluN and D-ManN is relatively low.

[0034] Example 5 Based on the property that (R)-L10 can specifically fluorescently recognize D-GalN from three D-amino sugars without being affected by other chiral amino acids and chiral sugars, we applied it to the analysis of D-amino sugar composition in different soil matrices. The D-amino sugar content in each sample accounted for 1% of the total mass of the silt sample. We extracted the soluble components from the three samples using ultrapure water, and after centrifugation and filtration, the supernatant was reacted with (R)-L10 for 3 hours for fluorescence signal analysis. The results were compared with those of laboratory-pure D-amino sugar aqueous solutions of the same concentration.

[0035] from Figure 9 It can be seen that different soil samples containing D-GalN emitted clearly distinguishable fluorescence signals after reacting with (R)-L10, and the fluorescence signal intensity of the three D-amino sugars in the soil extract ( Figure 9 (b)-(i)) and the same concentration of single D-amino sugar aqueous solution samples ( Figure 9 (a) The test results are very close; from Figure 10 It can be seen that the sludge sample contains D-GalN and D-GluN with different compositions. As the percentage of D-GalN in the sludge sample increases, the fluorescence signal exhibited by the probe also gradually increases. from Figure 11 It can be seen that the sludge sample contains D-GalN and D-ManN with different compositions. As the percentage of D-GalN in the sludge sample increases, the fluorescence signal exhibited by the probe gradually increases.

[0036] Example 6 This example demonstrates the fluorescent recognition of D-GalN in a biological matrix. HeLa cells were incubated with 20 μM (R)-L10 for 3 hours, then washed three times with PBS to remove excess probe, and then incubated with three different D-amino sugars for 3 hours each.

[0037] The results are as follows Figure 12 As shown: Cells with only the pure probe showed almost no obvious fluorescence signal; after adding the three D-amino sugars respectively, a very obvious bright yellow fluorescence signal was observed in cells with D-GalN, while only a very weak fluorescence signal was detected in cells with D-GluN and D-ManN.

[0038] The experimental conclusions of Examples 1-6 show that the fluorescent probe achieves specific fluorescent recognition of D-GalN based on the probe's chiral backbone, and can also determine the D-galactose content in soil and cells.

[0039] The above embodiments verified the selectivity, sensitivity, and practical application capability of probe (R)-L10 for D-GalN through time gradient, concentration gradient, soil extract, and cell experiments. The results are shown in the appendix. Figure 4 – Figure 12 .

[0040] The fluorescent probe (R)-L10 of this invention achieves specific fluorescent recognition of 0-30 equivalents of D-galactose, exhibits high detection stability, and can be used to determine the D-galactose content in soil and cells.

[0041] This invention successfully prepared a pair of novel chiral fluorescent probes (R)-L10 based on a binaphthyl skeleton. The synthetic route for these fluorescent probes is short and easy to operate. The aldehyde group of the fluorescent probe (R)-L10 can undergo an imine condensation reaction with the amino group of D-galactose. Furthermore, due to the differences in spatial configuration of different amino sugar molecules, the imine bond formed by D-galactose has the highest degree of conjugation with the naphthalene ring plane, thus producing a specific fluorescence enhancement effect.

[0042] The fluorescent probe of this invention achieves specific fluorescent recognition of 0-30 equivalents of D-galactose, exhibiting high detection stability and being unaffected by interference from other amino sugars, chiral amino acids, and chiral sugars. This fluorescent probe can be used for amino sugar composition analysis in soil. It can also be used for amino sugar imaging analysis in cells.

[0043] Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the present invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A highly sensitive and rapid detection and analysis method for amino sugars, characterized in that, Includes the following steps: S1, Synthetic fluorescent probe (R)-L10: S101. Weigh (R)-binaphthol and dissolve it in ultra-dry dichloromethane. Under 0°C conditions, slowly add N,N-diisopropylethylamine, then slowly add bromomethyl methyl ether. After the reaction is complete, add ultrapure water to quench the reaction, extract with dichloromethane, combine the organic phases, dry with anhydrous sodium sulfate, purify by column chromatography, and vacuum dry to obtain white intermediate (R)-6. S102. Weigh (R)-6 and dissolve it in ultra-dry tetrahydrofuran. Cool the solution to -78 °C and add n-butyllithium dropwise for 2 h. Then add ultra-dry N-N dimethylformamide dropwise. After the reaction is complete, add saturated ammonium chloride solution to quench the reaction under ice-water bath conditions. Extract with ethyl acetate, combine the organic phases, dry with anhydrous sodium sulfate, and purify by column chromatography to obtain intermediate (R)-7. S103. Weigh intermediate (R)-7 into a two-necked flask, evacuate and purge with nitrogen three times, add anhydrous acetonitrile to fully dissolve the raw material, then add naphthalimide fragment (8) and anhydrous K2CO3, heat under reflux at 80℃, after the reaction is complete, restore to room temperature, extract with dichloromethane, combine the organic phases, dry with anhydrous sodium sulfate, and purify by column chromatography to obtain yellow intermediate (R)-L9. S104. Weigh (R)-9, add dichloromethane and concentrated hydrochloric acid, stir thoroughly until the reaction is complete, then add sodium bicarbonate to adjust the pH to neutral, extract with dichloromethane, combine the organic phases, dry with anhydrous sodium sulfate, remove the solvent and obtain the fluorescent probe (R)-L10. S2. React the fluorescent probe (R)-L10 with the sample to be tested, and achieve specific recognition of D-galactosamine by detecting changes in fluorescence signal; The probe reacts with the sample to be tested, and the qualitative and quantitative analysis of D-GalN is achieved by detecting the change in fluorescence intensity at 550 nm. Specifically, the fluorescence detection of this invention is performed at 550 nm, and the excitation wavelength is 430 nm.

2. The method for high-sensitivity rapid detection and analysis of amino sugars according to claim 1, characterized in that, In step S101, the equivalent ratio of (R)-binaphthol, N,N-diisopropylethylamine and bromomethyl methyl ether is 1:2:1.

2.

3. The highly sensitive and rapid detection and analysis method for amino sugars according to claim 1, characterized in that, In step S101, the eluent used during silica gel column purification consists of petroleum ether and ethyl acetate in a volume ratio of 10:

1.

4. The highly sensitive and rapid detection and analysis method for amino sugars according to claim 1, characterized in that, In step S102, the amount of n-butyllithium is 1.5 equivalents and the amount of N,N dimethylformamide is 1.5 equivalents; the eluent used in silica gel column purification consists of petroleum ether and ethyl acetate in a volume ratio of 5:

1.

5. The method for high-sensitivity rapid detection and analysis of amino sugars according to claim 1, characterized in that, In step S103, the amount of anhydrous K2CO3 used is 2 equivalents; the amount of naphthalimide fragment (8) used is 1.5 equivalents; the eluent used in silica gel column purification is composed of petroleum ether and ethyl acetate in a volume ratio of 3:

1.

6. The highly sensitive and rapid detection and analysis method for amino sugars according to claim 1, characterized in that, In step S104, the concentrated hydrochloric acid is 10 equivalents; the eluent used in silica gel column purification consists of petroleum ether and ethyl acetate in a volume ratio of 1:1.

Images