A fluorescent probe for simultaneous detection of chiral acidic and basic amino acids, preparation, detection method and paper-based sensor thereof

Abstract

The application discloses a fluorescent probe for simultaneously detecting chiral acidic amino acids and basic amino acids, a preparation method, a detection method and a paper-based sensor thereof, and a specific chemical structural formula of the fluorescent probe is shown in the following formula: the fluorescent probe is simple in preparation and low in cost, and is high in detection sensitivity and good in selectivity for the acidic amino acids and the basic amino acids.

Description

Technical Field

[0001] This invention relates to the field of chemical analysis and detection technology, and in particular to a fluorescent probe for the simultaneous detection of chiral acidic and basic amino acids, its preparation, detection method, and its paper-based sensor. Background Technology

[0002] Amino acids are essential components of the human body and directly participate in physiological processes. Due to differences in their side chains, amino acids exhibit varying degrees of acidity and alkalinity, each playing a different role within the body. Acidic amino acids promote growth and development, maintain the immune system, and provide the nitrogen source necessary for growth. Alkaline amino acids aid in memory, learning, and sleep, are responsible for pain transmission and information processing, and provide the carbon source necessary for survival. Research has found that amino acids in the human body can be metabolized into products needed by the body through the assistance of various corresponding enzymes, achieving the interconversion between acidic and alkaline amino acids to ensure growth and development. This conversion relationship also exists in plant systems, where plants rely on assimilated carbon and synthesized amino acids to achieve optimal growth and development. However, the potential mechanisms and pathways of carbon and nitrogen conversion in different organs of animals and plants are not yet fully elucidated. Therefore, it is essential to develop a fluorescent probe capable of simultaneously recognizing glutamate and arginine for carbon and nitrogen monitoring in animals and plants, providing new research methods and ideas for studying carbon and nitrogen transport mechanisms. Summary of the Invention

[0003] The purpose of this invention is to provide a fluorescent probe for the simultaneous detection of chiral acidic amino acids and basic amino acids, its preparation method, detection method, and its paper-based sensor. The fluorescent probe is simple to prepare, low in cost, and has high sensitivity and good selectivity for the detection of acidic and basic amino acids.

[0004] The above-mentioned technical objective of the present invention is achieved through the following technical solution:

[0005] A fluorescent probe for simultaneously detecting chiral acidic and basic amino acids, characterized by the following specific chemical structural formula:

[0006]

[0007] This invention also provides a method for preparing a fluorescent probe for simultaneously detecting chiral acidic amino acids and basic amino acids, characterized by comprising the following steps:

[0008] S1. Weigh out S-binaphthol and sodium hydride and dissolve them separately in an appropriate amount of ultra-dry tetrahydrofuran. Mix the two solutions at -5 to 5°C and react for 1.5 to 2.5 hours. Then, restore the temperature to room temperature and continue the reaction. After 1.5 to 2.5 hours, cool the reaction system to -5 to 5°C and slowly add bromomethyl methyl ether. Then, restore the temperature to room temperature and continue the reaction. After the reaction is complete, quench the reaction with ultrapure water under ice-water bath conditions. Then, extract with ethyl acetate, combine the organic phases, dry with anhydrous sodium sulfate, and rotary evaporate to obtain a pale yellow solid crude product. Recrystallize and purify to obtain a white intermediate (R)-2.

[0009] S2. Weigh intermediate (R)-2 and dissolve it in an appropriate amount of ultra-dry tetrahydrofuran. Cool the solution to -78°C and add n-butyllithium dropwise. After restoring to room temperature, the reaction proceeds. After 0.5-1.5 hours, the solution becomes turbid. Then, cool the reaction solution to -5-5°C and gradually add ultra-dry N,N dimethylformamide. After restoring to room temperature, the reaction proceeds. After 1-3 hours, quench the reaction by adding saturated ammonium chloride solution under ice-water bath conditions. Extract with ethyl acetate, combine the organic phases, dry with anhydrous sodium sulfate, and rotary evaporate to obtain a yellow solid crude product. Purify by column chromatography and vacuum dry to obtain intermediate 2(R)-3.

[0010] S3. Weigh intermediate (R)-3 and dissolve it in an appropriate amount of dichloromethane solvent. Add trifluoroacetic acid. After 1-3 hours, add sodium bicarbonate to adjust the pH to neutral. Then extract with ethyl acetate, combine the organic phases, dry with anhydrous sodium sulfate, and rotary evaporate to obtain intermediate tri(R)-4.

[0011] S4. Weigh intermediate (R)-4 and dissolve it in an appropriate amount of dichloromethane solvent. Add 2-aminomethylpyridine and react at room temperature. After the reaction is complete, pour the reaction solution into cold methanol under ice-water bath conditions. Centrifuge and filter to obtain fluorescent probe powder.

[0012] More preferably, in step S1, the recrystallization solvent is methanol, the temperature is set to 70-90°C, and the molar ratio of S-binaphthol:sodium hydride:bromomethyl methyl ether is 1:6:3.

[0013] More preferably, in step S2, the volume ratio of the eluent phase for column chromatography purification is petroleum ether: ethyl acetate = 10:1, and the molar ratio of (R)-2: n-butyllithium: NN dimethylformamide is 2:6:5.

[0014] More preferably, the reactions in steps S1-S2 are all carried out under nitrogen protection.

[0015] The present invention also provides a method for detecting a fluorescent probe that can simultaneously detect chiral acidic amino acids and basic amino acids, characterized by comprising the following steps: dissolving the fluorescent probe in an appropriate amount of chromatographic grade dimethyl sulfoxide to prepare a concentrated probe solution, dissolving zinc acetate and amino acids separately in ultrapure water, and then sequentially adding dimethyl sulfoxide, probe solution, zinc acetate solution, and amino acid solution to a centrifuge tube, reacting for a period of time, and then adding a certain volume of dimethyl sulfoxide to dilute, and performing fluorescence testing.

[0016] More preferably, the molar ratio of the fluorescent probe: zinc acetate: amino acid is 1:1:1 to 40.

[0017] This invention also provides a paper-based sensor for simultaneously detecting chiral acidic and basic amino acids using fluorescent probes. The sensor is characterized by: dissolving an appropriate amount of fluorescent probe in methanol to prepare a solution of fluorescent probe, amino acid, and zinc acetate; adding the prepared amino acid solution and zinc acetate solution to the fluorescent probe solution for reaction; fully immersing glass fiber filter paper in the reaction solution; and air-drying the filter paper to obtain the test paper.

[0018] Further preferably, under natural light, the test paper reacting with arginine is orange-yellow, and the test paper reacting with other amino acids is yellow; under 365nm ultraviolet light, the test paper reacting with L-arginine is bright orange, and reacting with D-arginine is bright yellow; reacting with L-glutamic acid is dark green, and reacting with D-glutamic acid is bright green.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] Firstly, the fluorescent probe provided by this invention is simple to prepare and has low cost;

[0021] Secondly, the fluorescent probe of the present invention can simultaneously recognize acidic amino acids and basic amino acids from a variety of amino acids in dimethyl sulfoxide, and has good enantioselectivity for the two amino acids.

[0022] Thirdly, the detection method provided by this invention is simple to operate, uses inexpensive solvents, and has convenient post-processing.

[0023] Fourth, the paper-based sensor of the present invention can visually detect acidic and basic amino acids without requiring complex equipment. Attached Figure Description

[0024] Figure 1 The synthetic route for the fluorescent probe (R)-5 of this invention is shown below;

[0025] Figure 2 The synthetic route for the fluorescent probe (S)-5 of this invention is shown below;

[0026] Figure 3 The fluorescence spectra of the fluorescent probe (R)-5 of the present invention reacting with different concentrations of D-glutamic acid are shown.

[0027] Figure 4 The fluorescence spectra of the fluorescent probe (R)-5 of the present invention reacting with different concentrations of L-glutamic acid are shown.

[0028] Figure 5 The curve showing the relationship between the fluorescence intensity of the system at 540 nm and the concentration of glutamate is shown.

[0029] Figure 6 The fluorescence spectrum of the fluorescent probe (R)-5 of the present invention interacting with 8 eq D and L-glutamic acid;

[0030] Figure 7 The fluorescence spectrum of the fluorescent probe (R)-5 of the present invention interacting with 8 eq D and L-arginine;

[0031] Figure 8 The fluorescence spectrum of the fluorescent probe (R)-5 of this invention interacting with 39 amino acids (8 eq) is shown.

[0032] Figure 9 The fluorescence spectrum of the fluorescent probe (S)-5 of the present invention interacting with 8 eq D and L-glutamic acid;

[0033] Figure 10 The fluorescence spectrum of the fluorescent probe (S)-5 of the present invention interacting with 8 eq D and L-arginine;

[0034] Figure 11 The fluorescence spectrum of the fluorescent probe (S)-5 of the present invention interacting with 39 amino acids (8 eq);

[0035] Figure 12 The graph shows the color changes of (R)-5 probe paper reacting with different amino acids.

[0036] Figure 13 The color change graph shows the reaction of (S)-5 probe paper with different amino acids. Detailed Implementation

[0037] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the present invention.

[0038] Example, refer to Figure 1 Example 1: A method for preparing a fluorescent probe for simultaneous detection of chiral acidic and basic amino acids:

[0039] S1. Under a nitrogen atmosphere at 0°C, a tetrahydrofuran solution containing (R)-1 (11.5 g, 40.0 mmol) was slowly added dropwise to a tetrahydrofuran turbidity containing NaH (5.76 g, 240 mmol). (R)-1 was R-binaphthol. After the addition was complete, the reaction was allowed to proceed at room temperature for 2 hours. Subsequently, the reaction solution was cooled to 0°C, and bromomethyl methyl ether (10 mL, 120 mmol) was slowly added dropwise. The reaction was allowed to proceed at room temperature for 12 hours, at which point the solution turned into a milky white turbidity. The reaction was monitored by TLC plate to indicate completion. The resulting turbidity was poured into a cold saturated ammonium chloride solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated under vacuum, and then recrystallized from methanol to obtain a white powder (R)-2.

[0040] S2. Under a nitrogen atmosphere, (R)-2 (7.5 g, 20.0 mmol) was dissolved in 80 mL of tetrahydrofuran solution. The solution was cooled to -78 °C, and n-butyllithium (1.6 Mhexane solution, 37 mL, 60.1 mmol) was slowly added dropwise. After the addition was complete, the mixture was allowed to return to room temperature for 1 hour, during which time the solution became turbid. The reaction solution was then cooled to 0 °C, and N,N-dimethylformamide (3.9 mL, 50.1 mmol) was slowly added dropwise to the turbid solution. After the mixture was allowed to return to room temperature for 3 hours, the turbid solution turned orange. The reaction was monitored by TLC to indicate completion. The turbid solution was poured into a cold saturated ammonium chloride solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated under vacuum. The crude product was separated by thin-layer chromatography in petroleum ether and ethyl acetate solutions to obtain a yellow powder (R)-3.

[0041] S3. Add 1.0 mL of trifluoroacetic acid to a dichloromethane solution containing (R)-3 (1.00 g, 2.51 mmol). React at room temperature for 3 hours, until the solution turns purple-black. Monitor the reaction completion using a TLC plate. Pour the reaction solution into an aqueous solution, adjust the pH to approximately 7 with sodium bicarbonate, extract with ethyl acetate, dry to anhydrous sodium sulfate, and concentrate by vacuum evaporation to obtain an orange powder (R)-4.

[0042] S4. Add 0.7 mL of 2-aminomethylpyridine (6.43 mmol) to a dichloromethane solution containing (R)-4 (1.00 g, 2.92 mmol). React at room temperature for 6 hours, until the solution turns orange-red. The reaction is monitored by TLC to indicate completion. Pour the reaction solution into a cold methanol solution and centrifuge to obtain an orange-red powder (R)-5.

[0043] Example 2, refer to Figure 2 Using (S)-1 as a raw material, the enantiomer (S)-5 was synthesized using the same method as in the previous example.

[0044] S1. Under a nitrogen atmosphere at 0°C, a tetrahydrofuran solution containing (S)-1 (11.5 g, 40.0 mmol) was slowly added dropwise to a tetrahydrofuran turbidity containing NaH (5.76 g, 240 mmol). (S)-1 was S-binaphthol. After the addition was complete, the reaction was allowed to proceed at room temperature for 2 hours. Subsequently, the reaction solution was cooled to 0°C, and bromomethyl methyl ether (10 mL, 120 mmol) was slowly added dropwise. The reaction was allowed to proceed at room temperature for 12 hours, at which point the solution turned into a milky white turbidity. The reaction was monitored by TLC to indicate completion. The resulting turbidity was poured into a cold saturated ammonium chloride solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated under vacuum, and then recrystallized from methanol to obtain a white powder (S)-2.

[0045] S2. Under a nitrogen atmosphere, (S)-2 (7.5 g, 20.0 mmol) was dissolved in 80 mL of tetrahydrofuran solution. The solution was cooled to -78 °C, and n-butyllithium (1.6 Mhexane solution, 37 mL, 60.1 mmol) was slowly added dropwise. After the addition was complete, the mixture was allowed to return to room temperature for 1 hour, during which time the solution became turbid. The reaction solution was then cooled to 0 °C, and N,N-dimethylformamide (3.9 mL, 50.1 mmol) was slowly added dropwise to the turbid solution. After the mixture was allowed to return to room temperature for 3 hours, the turbid solution turned orange. The reaction was monitored by TLC to indicate completion. The turbid solution was poured into a cold saturated ammonium chloride solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated by vacuum evaporation. The crude product was separated by thin-layer chromatography in petroleum ether and ethyl acetate solutions to obtain a yellow powder (S)-3.

[0046] S3. Add 1.0 mL of trifluoroacetic acid to a dichloromethane solution containing (S)-3 (1.00 g, 2.51 mmol). React at room temperature for 3 hours, until the solution turns purple-black. Monitor the reaction completion using a TLC plate. Pour the reaction solution into an aqueous solution, adjust the pH to approximately 7 with sodium bicarbonate, extract with ethyl acetate, dry to anhydrous sodium sulfate, and concentrate by vacuum evaporation to obtain an orange powder (S)-4.

[0047] S4. Add 0.7 mL of 2-aminomethylpyridine (6.43 mmol) to a dichloromethane solution containing (S)-4 (1.00 g, 2.92 mmol). React at room temperature for 6 hours, until the solution turns orange-red. The reaction is monitored by TLC to indicate completion. Pour the reaction solution into a cold methanol solution and centrifuge to obtain an orange-red powder (S)-5.

[0048] (S)-5 is the chiral enantiomer of (R)-5, meaning its structure is a mirror image of (R)-5, but they cannot overlap. (S)-5 can be synthesized using (R)-1 as its enantiomer (S)-1. The bold lines in the diagram represent that they face outwards relative to the plane of the paper, and the products of S and R in the diagram have opposite orientations.

[0049] Example 3: A detection method for a fluorescent probe that simultaneously detects chiral acidic amino acids and basic amino acids:

[0050] Probe (R)-5 was dissolved in an appropriate amount of chromatographic grade dimethyl sulfoxide (DMSO) to prepare a 0.8 mM probe solution. Zinc acetate and amino acids were dissolved in ultrapure water to prepare 1 eq zinc acetate solution and 1-15 eq amino acid solution, respectively. Then, 250 μL of DMSO, 50 μL of probe solution, 50 μL of zinc acetate solution, and 50 μL of amino acid solution were added sequentially to centrifuge tubes, followed by dilution with 3.6 mL of DMSO. After reacting for 3 h, fluorescence was measured using the following parameters: excitation slit width 5 nm, emission slit width 5 nm, and excitation wavelength 420 nm. The fluorescent probe (R)-5 was reacted with different concentrations of D-glutamic acid, as described above. Figure 3 The fluorescent probe (R)-5 was reacted with different concentrations of L-glutamic acid, as per [reference needed]. Figure 4 The relationship between the fluorescence intensity of the system at 540 nm and the concentration of glutamate was tested, referring to... Figure 5 .

[0051] Reference Figure 6-8 In dimethyl sulfoxide, probe (R)-5 itself showed no obvious fluorescence. When (R)-5 reacted with 8 eq D-Glu and 1 eq zinc acetate, it produced a large fluorescence intensity at λ = 540 nm. The interaction of (R)-5 with 8 eq D-Arg also produced a large fluorescence intensity at 540 nm. When (R)-5 interacted with L-Glu and L-Arg, there was no significant increase in fluorescence intensity. When (R)-5 interacted with the other 17 amino acid enantiomers and glycine, the fluorescence intensity decreased significantly, and some interactions even resulted in no fluorescence response.

[0052] Reference Figure 9-11 Under the same conditions, (S)-5 showed a more significant fluorescence enhancement effect on D-Glu and D-Arg, with its fluorescence intensity being greater than that of D-Glu and D-Arg. The fluorescence responses of (R)-5 and (S)-5 to chiral arginine were mirror images of each other, indicating that (R)-5 has a chiral recognition effect on the two enantiomers of the amino acid.

[0053] Example 4: A paper-based sensor for simultaneously detecting chiral acidic and basic amino acids using fluorescent probes:

[0054] Weigh out an appropriate amount of (R)-5 and dissolve it in methanol to prepare a 12 mM probe solution. Then weigh out appropriate amounts of amino acids and zinc acetate to prepare amino acid solutions and zinc acetate solutions, respectively. Add the amino acid solutions and zinc acetate solutions to the probe solution and react for 10 minutes. Next, thoroughly soak glass fiber filter paper in the reaction solution, remove it, and air dry it naturally. The test paper will turn yellow or orange. Under natural light, the test paper reacting with arginine will be orange-yellow, while the test paper reacting with other amino acids will be yellow. (Reference) Figure 12 Under 365nm ultraviolet light, the test paper reacts with L-arginine to produce a bright orange color, and with D-arginine to produce a bright yellow color; it reacts with L-glutamic acid to produce a dark green color, and with D-glutamic acid to produce a bright green color.

[0055] The preparation process for the (S)-5 probe test strip is the same as that for the (R)-5 probe test strip. (Refer to...) Figure 13 The test results of the (S)-5 probe test strip are mirror images of those of the (R)-5 probe test strip.

[0056] The paper-based sensor of this invention can visually detect acidic and basic amino acids without requiring complex equipment.

[0057] The fluorescent probe of the present invention can simultaneously recognize acidic amino acids and basic amino acids from a variety of amino acids in dimethyl sulfoxide, and has good enantioselectivity for the two amino acids.

[0058] The detection method provided by this invention is simple to operate, uses inexpensive solvents, and has convenient post-processing.

[0059] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A fluorescent probe for simultaneously detecting chiral arginine and glutamate, characterized in that, The specific chemical structural formula is shown below: 。 2. The method for preparing a fluorescent probe for simultaneous detection of chiral arginine and glutamate according to claim 1, characterized in that, Includes the following steps: S1. Weigh R-binaphthol and sodium hydride and dissolve them separately in an appropriate amount of ultra-dry tetrahydrofuran. Mix the two solutions at -5 to 5°C and react for 1.5 to 2.5 hours. Then, restore the temperature to room temperature and continue the reaction. After 1.5 to 2.5 hours, cool the reaction system to -5 to 5°C, slowly add bromomethyl methyl ether, and restore the temperature to room temperature. After the reaction is complete, quench the reaction with ultrapure water under ice-water bath conditions, then extract with ethyl acetate. Combine the organic phases, dry with anhydrous sodium sulfate, and rotary evaporate to obtain a pale yellow solid crude product. Recrystallize and purify to obtain a white intermediate (R)-2. ; S2. Weigh intermediate (R)-2 and dissolve it in an appropriate amount of ultra-dry tetrahydrofuran. Cool the solution to -78 °C, add n-butyllithium dropwise, and allow the reaction to return to room temperature. After 0.5-1.5 h, the solution becomes turbid. Then cool the reaction solution to -5-5 °C, gradually add ultra-dry N,N-dimethylformamide, and allow the reaction to return to room temperature. After 1-3 h, quench the reaction with saturated ammonium chloride solution under ice-water bath conditions, extract with ethyl acetate, combine the organic phases, dry with anhydrous sodium sulfate, and rotary evaporate to obtain a yellow solid crude product. Purify by column chromatography and vacuum dry to obtain intermediate (R)-3. ; S3. Weigh intermediate (R)-3 and dissolve it in an appropriate amount of dichloromethane solvent. Add trifluoroacetic acid, and after 1-3 hours, add sodium bicarbonate to adjust the pH to neutral. Then extract with ethyl acetate, combine the organic phases, dry with anhydrous sodium sulfate, and rotary evaporate to obtain intermediate (R)-4. ; S4. Weigh intermediate (R)-4 and dissolve it in an appropriate amount of dichloromethane solvent. Add 2-aminomethylpyridine and react at room temperature. After the reaction is complete, pour the reaction solution into cold methanol under ice-water bath conditions. Centrifuge and filter to obtain fluorescent probe powder.

3. The method for preparing a fluorescent probe for simultaneous detection of chiral arginine and glutamate according to claim 2, characterized in that: In step S1, the recrystallization solvent is methanol, the temperature is set to 70-90°C, and the molar ratio of R-binaphthol:sodium hydride:bromomethyl methyl ether is 1:6:

3.

4. The method for preparing a fluorescent probe for simultaneous detection of chiral arginine and glutamate according to claim 2, characterized in that: In step S2, the volume ratio of the eluent phase for column chromatography purification is petroleum ether: ethyl acetate = 10:1, and the molar ratio of (R)-2: n-butyllithium: N,N-dimethylformamide is 2:6:

5.

5. The method for preparing a fluorescent probe for simultaneous detection of chiral arginine and glutamate according to claim 2, characterized in that: The reactions in steps S1-S2 were all carried out under nitrogen protection.

6. The detection method for non-disease diagnosis and treatment purposes using a fluorescent probe for simultaneous detection of chiral arginine and glutamate according to claim 1, characterized in that, Includes the following steps: The fluorescent probe was dissolved in an appropriate amount of chromatographic grade dimethyl sulfoxide to prepare a concentrated probe solution. Zinc acetate and amino acids were dissolved in ultrapure water, respectively. Then, dimethyl sulfoxide, probe solution, zinc acetate solution, and amino acid solution were added sequentially to a centrifuge tube. After reacting for a period of time, a certain volume of dimethyl sulfoxide was added to dilute the solution, and fluorescence testing was performed. The amino acid was arginine or glutamic acid.

7. The detection method for non-disease diagnosis and treatment purposes using a fluorescent probe for simultaneous detection of chiral arginine and glutamate according to claim 6, characterized in that: The molar ratio of the fluorescent probe, zinc acetate, and amino acid is 1:1:1 to 40, and the amino acid is arginine or glutamic acid.

8. A paper-based sensor for simultaneously detecting chiral arginine and glutamate using a fluorescent probe according to claim 1, characterized in that: Weigh an appropriate amount of fluorescent probe and dissolve it in methanol to prepare a fluorescent probe and zinc acetate solution. Add the prepared zinc acetate solution to the fluorescent probe solution to react. Soak glass fiber filter paper thoroughly in the reaction solution and let it air dry to obtain the test paper.

9. A paper-based sensor for simultaneously detecting chiral arginine and glutamate using a fluorescent probe according to claim 8, characterized in that: Under natural light, the test paper reacts with arginine to turn orange-yellow, and reacts with other amino acids to turn yellow. Under 365nm ultraviolet light, the test paper reacts with L-arginine to turn bright orange, reacts with D-arginine to turn bright yellow, reacts with L-glutamic acid to turn dark green, and reacts with D-glutamic acid to turn bright green.

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