A fluorescent probe for specific detection of D-alanine and its preparation method and detection method

Abstract

The present invention discloses a fluorescent probe for the specific detection of D-alanine, as well as its preparation and detection methods. The fluorescent probe has a BINOL backbone, and its structure is shown in Formula (1). By proposing a structure that can specifically recognize high concentrations of D-Ala in an aqueous phase, the fluorescent probe can specifically recognize D-Ala among the 20 amino acids in the human body, resulting in the probe having the advantages of specific selectivity, high sensitivity, and a wide detection concentration range.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent probes, and in particular to a fluorescent probe for specifically detecting D-alanine and a preparation method and a detection method thereof. Background Art

[0002] D-Ala is widely distributed in mammals. Limited data on D-Ala suggest its potential as a therapeutic agent for schizophrenia. D-Ala can be used as a drug to treat hyperactivity and other behavioral effects caused by pentachlorophenol. D-Ala also has protective effects against renal injury caused by ischemia-reperfusion. Furthermore, data suggest that D-Ala is an attractive therapeutic target and a potential biomarker for identifying acute kidney injury. Furthermore, blood levels of D-amino acids, including D-Ala, are reduced in severe viral infections, and D-alanine supplementation can improve prognosis. Therefore, D-Ala has great potential as a biomarker and treatment option for severe viral infections.

[0003] Currently, there are many methods for detecting D-Ala. Traditional methods include high-performance liquid chromatography, gas chromatography, and capillary electrophoresis. However, these methods suffer from expensive equipment, cumbersome procedures, and low sensitivity. Fluorescence sensing technology, by contrast, offers advantages such as high selectivity, high sensitivity, low detection limits, and ease of use. Using fluorescent probes to specifically identify D-Ala is a promising approach. Therefore, there is an urgent need for a fluorescent probe with unique selectivity, high sensitivity, and a wide detection concentration range for detecting D-Ala. Summary of the Invention

[0004] The present invention aims to provide a fluorescent probe for the specific detection of D-alanine, and a preparation method and detection method thereof. By proposing a structure of a fluorescent probe that can specifically recognize high concentrations of D-Ala in an aqueous phase, the fluorescent probe can specifically recognize D-Ala among the 20 amino acids in the human body, thereby having the advantages of specific selectivity, high sensitivity, and a wide detection concentration range.

[0005] The above technical objectives of the present invention are achieved through the following technical solutions:

[0006] A fluorescent probe for specific detection of D-alanine, wherein the fluorescent probe is based on BINOL as a skeleton, and its structure is shown in formula (1):

[0007] . (1)

[0009] The present invention also provides a method for preparing a fluorescent probe for specifically detecting D-alanine, comprising the following steps:

[0010]

[0011] Step 1, synthesis of (R)-2:

[0012] (R)-1 was dispersed in anhydrous THF and added to a solution of NaH dispersed in anhydrous THF at 0°C. The mixture was reacted at room temperature for 2 hours. Bromomethyl methyl ether was added at 0°C and the reaction was continued overnight at room temperature. H2O was added at 0°C to quench the reaction. The aqueous and organic phases were separated with ethyl acetate. After removing the solvent, the product was recrystallized to obtain a white solid product (R)-2.

[0013] Step 2, synthesis of (R)-3:

[0014] n-BuLi was added to a THF solution of (R)-2 at 0°C. After reacting for 1 hour, an appropriate amount of DMF was added at 0°C. After reacting at room temperature for 3 hours, a saturated ammonium chloride solution was added to quench the reaction. The aqueous and organic phases were separated, the solvent was removed under reduced pressure, and the product (R)-3 was purified by silica gel column chromatography to obtain a yellow product.

[0015] Step 3, synthesis of (R)-4:

[0016] (R)-3 was dissolved in a mixed solution of DCM and anhydrous ethanol, and an appropriate amount of HCl was added. The reaction was allowed to proceed overnight, and solid NaHCO3 was added to quench the reaction. DCM was added for extraction, and the aqueous and organic phases were separated. The solvent was removed under reduced pressure and the product (R)-4 was purified by silica gel column chromatography to obtain a yellow solid product.

[0017] Step 4, synthesis of (R)-5:

[0018] (R)-4 and hydroxylamine hydrochloride were dissolved in a mixture of DCM and anhydrous ethanol and stirred thoroughly. An appropriate amount of triethylamine was added, and the temperature was raised to 80°C, condensed and refluxed to react overnight. After sufficient reaction, the mixture was purified by silica gel column to obtain a light yellow solid (R)-5.

[0019] Further preferably, in step 2, the eluent used for silica gel column purification is petroleum ether: ethyl acetate = 10:1, v / v.

[0020] Further preferably, in step 4, the eluent used for silica gel column purification is petroleum ether:ethyl acetate = 10:1, v / v.

[0021] The present invention also provides a detection method for a fluorescent probe that specifically detects D-alanine. The fluorescent probe is dissolved in DMSO to prepare a 0.8 mM / L mother solution 1. D-alanine is dissolved in a diluted tetrabutylammonium hydroxide solution and reacted for 2 hours to prepare a 24 mM mother solution 2. Zinc acetate is dissolved in ultrapure water to prepare a 1.6 mM mother solution 3. 50 μL of solution is taken from each of mother solution 1 and mother solution 2 and added to a new sample bottle. 250 μL of acetonitrile is added. Subsequently, 50 μL of solution is taken from mother solution 3 and added to the sample bottle. The mixture is immediately mixed and reacted for three hours. After the reaction is completed, the volume is adjusted to 4 mL with acetonitrile.

[0022] In summary, the present invention has the following beneficial effects:

[0023] The present invention proposes a structure of a fluorescent probe that can specifically identify high-concentration D-Ala in the aqueous phase, so that the fluorescent probe can specifically identify D-Ala among the 20 amino acids in the human body, making it have the advantages of specific selectivity, high sensitivity, and a wide detection concentration range. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the synthetic route of the fluorescent probe in the present invention;

[0025] Figure 2 is the hydrogen nuclear magnetic resonance spectrum of (R)-5 of the present invention;

[0026] Figure 3 is the carbon NMR spectrum of (R)-5 of the present invention;

[0027] Figure 4 is the fluorescence spectrum of (R)-5 in the present invention for recognizing 20 amino acids;

[0028] Figure 5 This is a comparison of the fluorescence enhancement of (R)-5 in the present invention when recognizing 20 amino acids at 535.5 nm;

[0029] Figure 6 The fluorescence spectra of (R)-5 at different times after the reaction with D-Ala in the present invention;

[0030] Figure 7 is a graph of the fluorescence intensity at 535.5 nm at different times after the reaction of (R)-5 with D-Ala in the present invention;

[0031] Figure 8 The present invention is (R)-5 and 100 equivalents of D-Ala at different concentrations of Zn 2+ Fluorescence spectra of reactions in the system;

[0032] Figure 9is a graph of the fluorescence intensity at 535.5 nm after (R)-5 in the present invention reacts with different concentrations of D-Ala;

[0033] Figure 10 The present invention is (R)-5 and 100 equivalents of D-Ala at different concentrations of Zn 2+ Fluorescence spectra of reactions in the system;

[0034] Figure 11 The present invention is (R)-5 and 100 equivalents of D-Ala at different concentrations of Zn 2+ Fluorescence intensity at 535.5 nm after the reaction in the system. DETAILED DESCRIPTION

[0035] The present invention will be further described in detail below with reference to the accompanying drawings.

[0036] In the embodiment, a fluorescent probe for specifically detecting D-alanine is provided. The fluorescent probe is based on BINOL as a skeleton, and its structure is shown in formula (1):

[0037] . (1)

[0039] This compound is a trifunctional chiral fluorescent probe with BINOL as the skeleton, consisting of aldehyde, hydroxyl and hydroxylamine hydrochloride groups, which is used to quickly and specifically and selectively identify low-concentration D-Ala in acetonitrile.

[0040] This embodiment provides a method for preparing a fluorescent probe for specific detection of D-alanine. The synthetic route is as follows: Figure 1 The specific steps are as follows:

[0041] Step 1. Synthesis of (R)-2: (R)-1 was dispersed in anhydrous THF, added to a solution of NaH dispersed in anhydrous THF at 0°C, reacted at room temperature for 2 hours, added bromomethyl methyl ether at 0°C, reacted at room temperature overnight, quenched by adding H2O at 0°C, separated the aqueous and organic phases with ethyl acetate, and after removing the solvent, the product was recrystallized to obtain a white solid product (R)-2.

[0042] Step 2, synthesis of (R)-3: n-BuLi was added to a THF solution of (R)-2 at 0°C. After reacting for 1 hour, an appropriate amount of DMF was added at 0°C. After reacting at room temperature for 3 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. The product (R)-3 was purified by silica gel column purification using a petroleum ether:ethyl acetate ratio of 10:1, v / v, as the eluent.

[0043] Step 3. Synthesis of (R)-4: (R)-3 was dissolved in a mixed solution of DCM and anhydrous ethanol, and an appropriate amount of HCl was added. The reaction was allowed to proceed overnight, and solid NaHCO3 was added to quench the reaction. DCM was added for extraction, and the aqueous and organic phases were separated. The solvent was removed under reduced pressure and the product (R)-4 was purified by silica gel column to obtain a yellow solid product.

[0044] Step 4, Synthesis of (R)-5: (R)-4 and hydroxylamine hydrochloride were dissolved in a mixture of DCM and anhydrous ethanol and stirred thoroughly. An appropriate amount of triethylamine was added, and the mixture was heated to 80°C and refluxed for overnight reaction. After sufficient reaction, the mixture was purified on a silica gel column using petroleum ether:ethyl acetate = 10:1, v / v, as the eluent to obtain a pale yellow solid (R)-5.

[0045] like Figure 2 and Figure 3 As shown, (R)-5: 1 H NMR (400 MHz, DMSO-d6) δ 11.85 (s, 1H), 10.40 (s, 1H), 10.24 (s, 1H), 10.19 (s, 1H), 8.70 (s, 2H), 8.25 (s, 1H), 8.22 –8.15 (m, 1H), 8.00 (d, J = 8.8 Hz, 1H), 7.51 – 7.43 (m, 2H), 7.37 (dt, J =21.7, 6.7 Hz, 2H), 7.09 – 7.03 (m, 1H), 7.00 (d, J = 8.3 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 196.98, 153.65, 152.25, 151.24, 137.35, 136.81, 134.10,131.32, 130.68, 130.59, 129.06, 128.18, 127.78, 124.85, 124.61, 124.40,124.04, 123.46, 120.47, 117.48, 115.16.

[0046] (R)-5 specific selective test:

[0047] Pipette 50 μL of the mother solution from the (R)-5 standard sample and transfer it to a clean 5 mL centrifuge tube. Subsequently, pipette 50 μL of 24 mM (30 eq.) D-Ala into the centrifuge tube. At the same time, quickly pipette 250 μL of acetonitrile into the centrifuge tube, and immediately pipette 50 μL of zinc acetate solution to adjust the reaction volume to 400 μL. After gently shaking and mixing, let it stand at room temperature for 3 hours, and then adjust the reaction volume to 4 mL. After the dilution is completed, immediately transfer the solution to a cuvette for fluorescence spectroscopy testing, such as Figure 4 and Figure 5 shown.

[0048] (R)-5 Reaction Time Test:

[0049] Pipette 50 μL of mother liquor from the standard sample of (R)-5 and transfer it to a clean 5 mL centrifuge tube. Subsequently, pipette 50 μL of 24 mM (30 eq.) D-Ala and add it to the centrifuge tube. At the same time, quickly pipette 250 μL of acetonitrile, add it to the centrifuge tube, and immediately pipette 50 μL of zinc acetate solution to make the reaction solution dilute to 400 μL. After slight shaking to mix, react at room temperature for 1 min, 3 min, 5 min, 10 min, 20 min, 30 min, 40 min, 1 h, 2 h, 3 h, 4 h, 5 h, 6h, 7 h, 8 h, 9 h, 10 h, respectively. After the reaction is completed, the reaction solution is diluted to 4 mL. After the dilution is completed, the solution is immediately transferred to a cuvette for fluorescence spectroscopy test, such as Figure 6 and Figure 7 shown.

[0050] D-Ala equivalent test of (R)-5

[0051] Pipette 50 μL of the mother solution from the standard sample of (R)-5 and transfer it to a clean 5 mL centrifuge tube. Subsequently, take 50 μL of 0.8 mM, 1.6 mM, 3.2 mM, 4.8 mM, 8 mM, 10.4 mM, 12.8 mM, 16 mM, 17.6 mM, 19.2mM, 20.8 mM, 24 mM, 25.6 mM, 27.2 mM, 28.8 mM and 32 mM D-Ala and add them to the centrifuge tube. At the same time, quickly draw 250 μL of acetonitrile and add it to the centrifuge tube, and immediately draw 50 μL of zinc acetate solution to make the reaction solution 400 μL. After slight shaking to mix, let it stand at room temperature for 3 hours, and then make the reaction solution 4 mL. After the dilution is completed, immediately transfer the solution to a cuvette for fluorescence spectroscopy test, such as Figure 8 and Figure 9 shown.

[0052] Zn 2+ Test of the effect of concentration on fluorescence intensity:

[0053] Pipette 50 μL of the mother liquor from the (R)-5 standard sample and transfer it to a clean 5 mL centrifuge tube. Subsequently, pipette 50 μL of 24 mM (30 equivalents) D-Ala into the centrifuge tube. At the same time, pipette 250 μL of acetonitrile, add it to the centrifuge tube, and immediately pipette 50 μL of 0.4 mM, 0.8 mM, 1.2 mM, 1.6 mM, 2.0 mM, 2.4 mM, and 3.2 mM zinc acetate solutions to make the reaction solution 400 μL. After gently shaking to mix, let it stand at room temperature for 3 hours, and then make the reaction solution 4 mL. After the dilution is completed, immediately transfer the solution to a cuvette for fluorescence spectroscopy testing, such as Figure 10 and Figure 11 shown.

[0054] In summary, (R)-5 was introduced with 22 equivalents of 20 amino acids (19 of which were chiral) to test its selectivity for specific fluorescence recognition, with D-Ala showing the best performance. In acetonitrile, (R)-5 exhibited chemoselective fluorescence enhancement for 22 equivalents of D-Ala, with detection completed in three hours. The duration of probe recognition for D-Ala was tested at reaction times of 1 min, 3 min, 5 min, 10 min, 20 min, 30 min, 40 min, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, and 10 h. Results showed that fluorescence enhancement reached the ideal intensity after 3 h.

[0055] The fluorescence enhancement of (R)-5 to D-Ala showed a trend of first increasing and then decreasing in the range of 1-40 equivalents.

[0056] Zn 2+ The concentration of Zn has a certain effect on the recognition of D-Ala by the probe. 2+ When the amount is higher than 3 equivalents, the fluorescence intensity of L-Ala will decrease with the increase of Zn 2+ It is enhanced with the increase of concentration.

[0057] The present invention also provides a method for detecting a fluorescent probe that specifically detects D-alanine. The detection method is as follows:

[0058] The fluorescent probe was dissolved in DMSO to prepare a 0.8 mM / L stock solution 1. D-alanine was dissolved in a diluted tetrabutylammonium hydroxide solution and reacted for 2 hours to prepare a 24 mM stock solution 2. Zinc acetate was dissolved in ultrapure water to prepare a 1.6 mM stock solution 3. 50 μL of each of stock solutions 1 and 2 were added to a new sample vial. 250 μL of acetonitrile was then added. Then, 50 μL of stock solution 3 was added to the sample vial. Immediately mix thoroughly and react for three hours. After the reaction, the volume was adjusted to 4 mL with acetonitrile.

[0059] This specific embodiment is merely an explanation of the present invention and is not intended to limit the present invention. After reading this specification, those skilled in the art may make non-creative modifications to this embodiment as needed. However, as long as such modifications are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. A fluorescent probe for specific detection of D-alanine, characterized in that: The fluorescent probe is based on BINOL as the skeleton, and its structure is shown in formula (1): .

2. The method for preparing a fluorescent probe for specific detection of D-alanine according to claim 1, wherein: The following steps are involved: Step 1, synthesis of (R)-2: (R)-1 was dispersed in anhydrous THF, and a solution of NaH dispersed in anhydrous THF was added at 0°C, and the mixture was reacted at room temperature for 2 hours. Bromomethyl methyl ether was added at 0°C, and the mixture was reacted at room temperature overnight. H2O was added at 0°C to quench the reaction, and the aqueous and organic phases were separated with ethyl acetate. After removing the solvent, the product was recrystallized to obtain a white solid product (R)-2; Step 2, synthesis of (R)-3: n-BuLi was added to a THF solution of (R)-2 at 0°C. After reacting for 1 hour, an appropriate amount of DMF was added at 0°C. After reacting at room temperature for 3 hours, a saturated ammonium chloride solution was added to quench the reaction. The aqueous and organic phases were separated, the solvent was removed under reduced pressure, and the product (R)-3 was purified by silica gel column chromatography to obtain a yellow product. Step 3, synthesis of (R)-4: (R)-3 was dissolved in a mixed solution of DCM and anhydrous ethanol, and an appropriate amount of HCl was added. The reaction was allowed to proceed overnight, and solid NaHCO3 was added to quench the reaction. DCM was added for extraction, and the aqueous and organic phases were separated. The solvent was removed under reduced pressure and the product (R)-4 was purified by silica gel column chromatography to obtain a yellow solid product. Step 4, synthesis of (R)-5: (R)-4 and hydroxylamine hydrochloride were dissolved in a mixture of DCM and anhydrous ethanol and stirred thoroughly. An appropriate amount of triethylamine was added, and the temperature was raised to 80°C, condensed and refluxed to react overnight. After sufficient reaction, the mixture was purified by silica gel column to obtain a light yellow solid (R)-5.

3. The method for preparing a fluorescent probe for specific detection of D-alanine according to claim 2, wherein: In step 2, the eluent used for silica gel column purification was petroleum ether:ethyl acetate = 10:1, v / v.

4. The method for preparing a fluorescent probe for specific detection of D-alanine according to claim 3, wherein: In step 4, the eluent used for silica gel column purification was petroleum ether:ethyl acetate = 10:1, v / v.

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