A compound, a probe, a preparation method and an application thereof for identifying and distinguishing basic amino acids in an aqueous phase

Abstract

The present invention provides a compound, a probe, a preparation method and an application for identifying and distinguishing basic amino acids in an aqueous phase, wherein the preparation method comprises: reacting binaphthol and sodium hydride, then adding bromomethyl methyl ether to react to obtain an intermediate compound 2; reacting compound 2 with n-butyl lithium, then adding N-N dimethylformamide to react to obtain compound 3; reacting compound 3 with concentrated hydrochloric acid to obtain compound 4; reacting compound 4 with triethylamine to obtain a target compound. The preparation method of the present invention is simple to synthesize, low in price, moderate in reaction conditions, high in recognition sensitivity, convenient in post-processing, and very friendly to the environment; the compound obtained by the preparation method can be used to prepare a fluorescent probe, and the detection method has a short response time, good selectivity, and a high degree of visualization by using the principle of hydrogen bond reaction, which meets the needs of rapid detection of basic amino acids.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical engineering, and in particular relates to a compound, a probe, a preparation method and an application thereof for identifying and distinguishing basic amino acids in an aqueous phase. Background Art

[0002] Amino acids are the basic substances that make up proteins and are indispensable nutrients in organisms. There are about 300 natural amino acids that have been discovered, and there are 20 common amino acids that make up biological proteins. Amino acids can be divided into acidic, neutral and basic amino acids due to the different acidity and alkalinity of the groups on the side chains. Among them, basic amino acids are amino acids with two amino groups and one carboxyl group. Their characteristics are that the side chains often contain protonable basic chemical groups, namely guanidine, amino and imidazole groups. Basic amino acids include arginine, lysine and histidine. Arginine (Arg), as a semi-essential amino acid for the human body, plays a vital role in many chemical and biological applications, such as molecular transporters, photocatalysts, supramolecular nanofibers, nanostructures, protein biopharmaceuticals, and the direct formation of NO, urea, and ornithine. In addition, arginine has a significant effect on human brain chemistry and is also involved in important life activities such as vasodilation, immune response and neurotransmission.

[0003] Lysine (Lys) is an essential amino acid for the human body. It is closely involved in the ornithine cycle and polyamine synthesis. It is decomposed in the liver of mammals through the saccharin pathway. High levels of lysine in plasma and urine indicate innate metabolic disorders such as cystinuria or hyperlysinemia. In addition, lysine cannot be synthesized in the human body and can only be taken from the outside. Therefore, lysine content is usually proposed as an evaluation criterion for evaluating the nutritional value of food. Therefore, the detection of lysine has a positive nutritional signal.

[0004] Histidine (His), as an essential amino acid for infants, can not only serve as a neurotransmitter or neuromodulator in human muscles and nervous systems, but also assist in the transfer of metal elements due to its unique imidazole group. Medical studies have shown that persistent deficiency of histidine can lead to chronic kidney disease, rheumatoid arthritis and other functional disorders, while abnormally elevated concentrations can be associated with asthma and cirrhosis. In addition, histidine can be used as a tumor marker for renal cell carcinoma and is used to monitor renal cell carcinoma.

[0005] In current detection technologies, only three basic amino acids can be detected simultaneously, but they cannot be distinguished. Each basic amino acid has different importance. How to accurately identify and distinguish these three basic amino acids has a more profound significance. In addition, amino acids often play a role in the aqueous phase, while many current detection methods are in the organic phase. Identifying amino acids in the aqueous phase is one of the current difficulties. Therefore, detection technology that can identify and distinguish basic amino acids in the aqueous phase needs to be developed urgently. Summary of the invention

[0006] The purpose of the present invention is to overcome the above-mentioned problems existing in the conventional technology and to provide a compound, a probe, a preparation method and an application thereof for identifying and distinguishing basic amino acids in an aqueous phase.

[0007] In order to achieve the above technical objectives and the above technical effects, the present invention is implemented through the following technical solutions:

[0008] A method for preparing a compound for identifying and distinguishing basic amino acids in an aqueous phase comprises the following steps:

[0009] S1: Weigh binaphthol and sodium hydride and dissolve them in an appropriate amount of ultra-dry tetrahydrofuran respectively. Under appropriate temperature conditions, mix the two solutions evenly and then return to room temperature for reaction. After a certain period of time, cool the reaction system to an appropriate temperature, then add bromomethyl methyl ether dropwise, then return to room temperature for reaction overnight. Add ultrapure water in an ice-water bath to quench the reaction, then extract with ethyl acetate, combine the organic phases, dry over anhydrous sodium sulfate, and rotary evaporate to obtain a light yellow solid product, which is purified by recrystallization solvent to obtain a white intermediate compound 2.

[0010] S2: Weigh compound 2 and dissolve it in an appropriate amount of ultra-dry tetrahydrofuran. Under appropriate temperature conditions, add n-butyl lithium dropwise, the reaction system changes from colorless to brown, and returns to room temperature for reaction; after a certain period of time, cool the reaction system to an appropriate temperature, and then gradually add ultra-dry NN dimethylformamide, the reaction system changes to turbid light blue, and returns to room temperature for reaction; after a certain period of time, add saturated ammonium chloride solution in an ice-water bath to quench the reaction, then extract with ethyl acetate, combine the organic phases, dry over anhydrous magnesium sulfate, and rotary evaporate to obtain a yellow solid product, purify by column chromatography, and vacuum dry to obtain compound 3;

[0011] S3: Compound 3 was dissolved in a proper amount of a mixed solvent of dichloromethane and anhydrous ethanol, and concentrated hydrochloric acid was added dropwise; after a certain period of time, sodium bicarbonate was added to quench the reaction, and then extracted with dichloromethane, the organic phases were combined, dried over anhydrous magnesium sulfate, and rotary evaporated to obtain a yellow solid compound 4;

[0012] S4: Weigh compound 4 and dissolve it in a proper amount of a mixed solvent of dichloromethane and anhydrous methanol, add a proper amount of triethylamine, react for a certain period of time under appropriate temperature conditions, then return to room temperature, dry over anhydrous magnesium sulfate, and rotary evaporate to obtain a yellow solid product, purify by column chromatography, and vacuum dry to obtain the target compound.

[0013] Further, in the method for preparing a compound for identifying and distinguishing basic amino acids in an aqueous phase as described above, in step S1, the suitable temperature is -5 to 5°C; the certain time is 1.5 to 2.5 hours; the recrystallization solvent is methanol, and the temperature is set to 70 to 90°C;

[0014] In step S2, the suitable temperature is -5 to 5°C;

[0015] When the reaction system changes from colorless to brown and returns to room temperature, the reaction time is 0.5h to 1.5h;

[0016] The reaction system turns into a turbid light blue and returns to room temperature for 2.5 to 3.5 hours.

[0017] The volume ratio of the elution phase in the column chromatography purification is petroleum ether:ethyl acetate=10:1;

[0018] The molar ratio of (R)-3: n-butyl lithium: NN dimethylformamide is 1:2.5:2.5.

[0019] Further, in the method for preparing a compound capable of identifying and distinguishing basic amino acids in an aqueous phase as described above, in step S3, the certain time is overnight;

[0020] The volume ratio of dichloromethane to anhydrous methanol is 1:1.

[0021] Further, in the method for preparing a compound capable of identifying and distinguishing basic amino acids in an aqueous phase as described above, in step S4:

[0022] The suitable temperature is 60-80°C;

[0023] The certain time is 8 to 12 hours;

[0024] The volume ratio of the elution phase in the column chromatography purification is petroleum ether:ethyl acetate=5:1;

[0025] The molar ratio of the compound 4: hydroxylamine hydrochloride: triethylamine is 1:4:8.

[0026] Furthermore, in the method for preparing the compound capable of identifying and distinguishing basic amino acids in an aqueous phase as described above, the binaphthol is (R)-BINOL; and the molar ratio of the binaphthol: sodium hydride: bromomethyl methyl ether is 1:6:3.

[0027] The compound capable of recognizing and distinguishing basic amino acids in the aqueous phase prepared by the preparation method described above.

[0028] A fluorescent probe for recognizing and distinguishing basic amino acids in an aqueous phase comprises the compound as described above.

[0029] An application of the compound as described above to recognize and distinguish basic amino acids in an aqueous phase.

[0030] Applications as described above, including:

[0031] Preparing a probe solution: dissolving the compound in an appropriate amount of chromatography-grade tetrahydrofuran to obtain the probe solution;

[0032] Dissolve zinc acetate and amino acid in ultrapure water to prepare 3 eq zinc acetate solution and 0.5-10 eq amino acid solution respectively;

[0033] Add amino acid solution, zinc acetate solution, and probe solution to a centrifuge tube in sequence, and then add ultrapure water to dilute; after reacting for 20 minutes, perform fluorescence test, wherein the fluorescence test parameters are: excitation light slit is 5nm, emission light slit is 5nm, and excitation light wavelength is 390nm;

[0034] Identify and differentiate basic amino acids through fluorescence test results and colorimetric solution effects.

[0035] Applications as described above, including:

[0036] Preparing a probe solution: dissolving the compound in an appropriate amount of tetrahydrofuran to obtain the probe solution;

[0037] Add 10eq amino acid and 3eq zinc acetate solution into a clean reaction centrifuge tube;

[0038] The probe solution was added to the centrifuge tube and reacted at room temperature for 5 min;

[0039] Basic amino acids can be identified and differentiated by observing the color of the reaction solution under 365nm ultraviolet light.

[0040] The beneficial effects of the present invention are:

[0041] The present invention provides a compound, a probe, a preparation method and an application for identifying and distinguishing basic amino acids in an aqueous phase. The preparation method is simple to synthesize, low in price, moderate in reaction conditions, high in recognition sensitivity, convenient in post-processing and very environmentally friendly. The fluorescent probe prepared by the compound uses the hydrogen bond reaction principle, and the detection method has a short response time, good selectivity and high visualization, which meets the needs of rapid detection of basic amino acids and has important application value in many fields.

[0042] Of course, any product implementing the present invention does not necessarily need to achieve all of the above advantages at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for describing the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0044] Figure 1 This is the synthetic route of the compound in Example 1 of the present invention;

[0045] Figure 2 The fluorescence spectra of the compound of the present invention acting on arginine at different concentrations;

[0046] Figure 3 This is the relationship curve between the fluorescence intensity of the system at 540nm and the arginine concentration;

[0047] Figure 4 The fluorescence spectra of the compound of the present invention acting on lysine at different concentrations;

[0048] Figure 5 This is the relationship curve between the fluorescence intensity of the system at 540nm and the lysine concentration;

[0049] Figure 6 The fluorescence spectrum of the compound of the present invention acting on 10 eq arginine and 10 eq lysine;

[0050] Figure 7 It is a fluorescence intensity diagram of the compound of the present invention and 20 kinds of amino acids (10eq) at 540nm;

[0051] Figure 8 The fluorescence spectra of the compound of the present invention and 20 kinds of amino acids (10eq) are shown;

[0052] Fig. 9 The color diagram of the detection solution of the probe reacting with three basic amino acids under 365nm ultraviolet light;

[0053] Fig.10 are the chemical structural formulas of three basic amino acids;

[0054] Fig.11 is a reaction mechanism diagram of the compound of the present invention and three basic amino acids;

[0055] Fig.12 is the H NMR spectrum of (R)-1;

[0056] Fig.13 is the H NMR spectrum of (R)-2;

[0057] Fig.14 , 15 The H-NMR spectrum and C-NMR spectrum of (R)-3;

[0058] Fig.16 , 17 The H-NMR spectrum and C-NMR spectrum of (R)-4;

[0059] Fig.18 It is the mass spectrum data diagram of compound (R)-4 of the present invention. DETAILED DESCRIPTION

[0060] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0061] Example 1

[0062] This embodiment provides a method for preparing a compound that recognizes arginine or lysine, such as Figure 1 As shown, the following steps are included:

[0063] S1. Weigh (R)-BINOL (18mmol, 5g) and sodium hydride (108mmol, 4.3g) and dissolve them in 30mL ultra-dry tetrahydrofuran, mix the two solutions at 0℃, and return to room temperature for reaction after completion. After 2h, cool the reaction system to 0℃, add bromomethyl methyl ether (54mmol, 4.4mL) dropwise, and then return to room temperature for reaction overnight. Add ultrapure water to quench the reaction under ice-water bath conditions, extract with ethyl acetate, combine the organic phases, dry over anhydrous sodium sulfate, and rotary evaporate to obtain a pale yellow solid crude product, purify with methanol, set the temperature to 80℃, and vacuum dry to obtain a white solid (R)-1.

[0064]

[0065] S2. Weigh (R)-1 (10.68mmol, 4g) and dissolve it in 45mL of ultra-dry tetrahydrofuran. At 0℃, add n-butyl lithium (26.7mmol, 16.6mL) dropwise. The reaction system changes from colorless to brown and returns to room temperature for reaction. After 1h, cool the reaction system to 0℃, and then gradually add ultra-dry NN dimethylformamide (25.64mmol, 2mL). The system changes to turbid light blue and returns to room temperature for reaction. After 3h, add saturated ammonium chloride solution in an ice-water bath to quench the reaction, extract with ethyl acetate, combine the organic phases, dry over anhydrous sodium sulfate, and rotary evaporate to obtain a yellow solid crude product, which is purified by column chromatography. The volume ratio of the elution phase for column chromatography purification is petroleum ether: ethyl acetate = 10:1, and vacuum dry to obtain (R)-2.

[0066]

[0067] S3, (R)-2 was dissolved in a mixed solvent of 10 mL of dichloromethane and 10 mL of anhydrous ethanol, and 1 ml of concentrated hydrochloric acid was added dropwise. The reaction was allowed to proceed overnight, and sodium bicarbonate was added to quench the reaction, and then the mixture was extracted with dichloromethane, and the organic phases were combined, dried over anhydrous sodium sulfate, and rotary evaporated to obtain a yellow solid (R)-3.

[0068]

[0069] S4. Weigh (R)-3 (1 mmol, 342 mg) and hydroxylamine hydrochloride (4 mmol, 278 mg) and dissolve them in a mixed solution of 20 mL of dichloromethane and 20 mL of methanol, then gradually add triethylamine (8 mmol, 1.12 mL). After completion, heat the reaction to 65 °C and heat under reflux overnight. After the reaction is restored to room temperature, dry with anhydrous magnesium sulfate, and rotary evaporate to obtain a yellow solid crude product. Purify by column chromatography, wherein the volume ratio of the elution phase for column chromatography purification is petroleum ether: ethyl acetate = 5:1, and vacuum dry to obtain the fluorescent probe (R)-4.

[0070]

[0071] The preparation method provided by the embodiment of the present invention is simple, low in cost and has mild reaction conditions.

[0072] Example 2

[0073] The method for identifying and distinguishing basic amino acids in an aqueous phase using the compound prepared in Example 1 comprises:

[0074] The probe (R)-4 was dissolved in an appropriate amount of chromatographic grade tetrahydrofuran to prepare a 0.8mM probe solution, and zinc acetate and amino acid were dissolved in ultrapure water to prepare 3eq zinc acetate solution and 0.5-10eq amino acid solution. Then, 50uL amino acid solution, 50uL zinc acetate solution, and 50uL probe solution were added to the centrifuge tube in sequence, and then 3.85ml ultrapure water was added for dilution. After reacting for 20 minutes, a fluorescence test was performed, wherein the fluorescence test parameters were: the excitation light slit was 5nm, the emission light slit was 5nm, and the excitation light wavelength was 390nm.

[0075] like Figure 2-Figure 8 As shown, (R)-4 itself has no obvious fluorescence in the aqueous phase. When (R)-4 reacts with 10 eqArg and 3 eq zinc acetate, a larger fluorescence intensity is generated at λ=540nm. When (R)-4 reacts with 10 eqLys and 3 eq zinc acetate, the fluorescence intensity generated at λ=540nm is slightly weaker than that of arginine. When (R)-4 interacts with the other 18 amino acids, the fluorescence intensity is significantly reduced, and most of them even have no fluorescence response.

[0076] The compound prepared by the preparation method of the present invention can specifically recognize arginine or lysine among various amino acids in an aqueous phase.

[0077] Example 3

[0078] Applications of compound-based colorimetric detection solutions:

[0079] like Fig. 9 As shown, 1 mL of 10 eq amino acid and 1 mL of 3 eq zinc acetate were added to a 5 mL cuvette and mixed for 1 min, then 1 mL of the probe solution was added thereto. After reacting for 5 min, the mixture was irradiated with a 365 nm ultraviolet lamp, wherein the arginine detection solution was bright yellow, lysine was fluorescent green, and histidine was transparent.

[0080] The detection solution of the embodiment of the present invention has the advantages of short response time, good selectivity and high visualization, and meets the needs of rapid identification and differentiation of three basic amino acids.

[0081] The detection method provided in the embodiment of the present invention adopts a green solvent, which is low in price, convenient for post-processing, and very friendly to the environment.

[0082] The preferred embodiments of the present invention disclosed above are only used to help explain the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to specific implementation methods. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A method for preparing a compound for recognizing and distinguishing basic amino acids in an aqueous phase, characterized in that: The steps include: S1: Weigh binaphthol and sodium hydride and dissolve them in an appropriate amount of ultra-dry tetrahydrofuran. Mix the two solutions evenly under appropriate temperature conditions, then return to room temperature for reaction. After a certain period of time, cool the reaction system to an appropriate temperature, then add bromomethyl methyl ether dropwise, then return to room temperature for reaction overnight. Add ultrapure water in an ice-water bath to quench the reaction, then extract with ethyl acetate, combine the organic phases, dry over anhydrous sodium sulfate, and rotary evaporate to obtain a light yellow solid product, which is then purified by recrystallization solvent to obtain a white intermediate compound 2. The binaphthol is (R)-BINOL; the molar ratio of the binaphthol: sodium hydride: bromomethyl methyl ether is 1:6:3; The structural formula of compound 2 is: ; S2: Compound 2 was weighed and dissolved in an appropriate amount of ultra-dry tetrahydrofuran. Under appropriate temperature conditions, n-butyl lithium was added dropwise, and the reaction system changed from colorless to brown, and then returned to room temperature for reaction; after a certain period of time, the reaction system was cooled to an appropriate temperature, and ultra-dry NN dimethylformamide was gradually added, and the reaction system turned into a turbid light blue, and returned to room temperature for reaction; after a certain period of time, a saturated ammonium chloride solution was added in an ice-water bath to quench the reaction, and then extracted with ethyl acetate, the organic phases were combined, dried over anhydrous magnesium sulfate, and rotary evaporated to obtain a yellow solid product, which was purified by column chromatography and dried in vacuo to obtain compound 3; The structural formula of compound 3 is: ; S3: Compound 3 was dissolved in a suitable amount of a mixed solvent of dichloromethane and anhydrous ethanol, and concentrated hydrochloric acid was added dropwise. After a certain period of time, sodium bicarbonate was added to quench the reaction, and the mixture was extracted with dichloromethane. The organic phases were combined, dried over anhydrous magnesium sulfate, and rotary evaporated to obtain a yellow solid compound 4. The structural formula of compound 4 is: ; S4: Weigh compound 4 and hydroxylamine hydrochloride and dissolve them together in a suitable amount of a mixed solvent of dichloromethane and anhydrous methanol. Add an appropriate amount of triethylamine dropwise and react for a certain period of time at a suitable temperature. Then return the mixture to room temperature, dry over anhydrous magnesium sulfate, and rotary evaporate to obtain a yellow solid product. Purify it by column chromatography and dry it in vacuo to obtain the target compound. The structural formula of the target compound is: .

2. The method for preparing a compound capable of recognizing and distinguishing basic amino acids in an aqueous phase according to claim 1, wherein: In step S1, the suitable temperature is -5 to 5°C; the certain time is 1.5 to 2.5 hours; the recrystallization solvent is methanol, and the temperature is set to 70 to 90°C; In step S2, the suitable temperature is -5 to 5°C; When the reaction system changes from colorless to brown and returns to room temperature, the reaction time is 0.5h to 1.5h; The reaction system turns turbid light blue and returns to room temperature for 2.5 to 3.5 hours. The volume ratio of the elution phase for column chromatography purification is petroleum ether:ethyl acetate=10:1; The molar ratio of the compound 2: n-butyl lithium: N-N-dimethylformamide is 1:2.5:2.

5.

3. The method for preparing a compound capable of recognizing and distinguishing basic amino acids in an aqueous phase according to claim 1, wherein: In step S3, the certain time is overnight; The volume ratio of dichloromethane to anhydrous methanol is 1:

1.

4. The method for preparing a compound capable of recognizing and distinguishing basic amino acids in an aqueous phase according to claim 1, wherein: In step S4, the suitable temperature is 60-80°C; The certain time is 8 to 12 hours; The volume ratio of the elution phase for column chromatography purification is petroleum ether:ethyl acetate = 5:1; The molar ratio of the compound 4: hydroxylamine hydrochloride: triethylamine is 1:4:

8.

5. A compound capable of recognizing and distinguishing basic amino acids in an aqueous phase prepared by the preparation method according to claim 1.

6. A fluorescent probe for identifying and distinguishing basic amino acids in aqueous phase, characterized in that: Including the compound as claimed in claim 5.

7. Use of the compound according to claim 5 for identifying and distinguishing basic amino acids in an aqueous phase.

8. The use according to claim 7, characterized in that include: preparing a probe solution: dissolving the compound in an appropriate amount of chromatography-grade tetrahydrofuran to obtain the probe solution; Dissolve zinc acetate and amino acid in ultrapure water to prepare 3 eq zinc acetate solution and 0.5-10 eq amino acid solution respectively; An amino acid solution, a zinc acetate solution, and a probe solution were sequentially added to a centrifuge tube, and then diluted with ultrapure water. After reacting for 20 minutes, a fluorescence test was performed, wherein the fluorescence test parameters were: an excitation light slit of 5 nm, an emission light slit of 5 nm, and an excitation light wavelength of 390 nm. Identify and differentiate basic amino acids through fluorescence test results and colorimetric solution effects.

9. The use according to claim 7, characterized in that include: preparing a probe solution: dissolving the compound in an appropriate amount of tetrahydrofuran to obtain the probe solution; Add 10eq of amino acid and 3eq of zinc acetate solution into a clean reaction centrifuge tube; The probe solution was added to the centrifuge tube and reacted at room temperature for 5 min; Basic amino acids can be identified and differentiated by observing the color of the reaction solution under 365nm ultraviolet light.

Images