A fluorescent probe for detecting L-tyrosine (L-Tyr) and a preparation method and a detection method thereof

Abstract

The application discloses a fluorescent probe for detecting L-tyrosine (L-Tyr) and a preparation method and a detection method thereof, and a specific chemical structural formula of the fluorescent probe is shown in the following formula: the fluorescent probe is simple in preparation, low in cost, high in detection sensitivity and good in selectivity.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of chemical analysis and detection technology, in particular to a fluorescent probe for detecting L-tyrosine (L-Tyr), and a preparation method and a detection method thereof. The probe has advantages of specific selectivity, high sensitivity, wide detection concentration range, etc. BACKGROUND

[0002] Amino acids are an important component of the human body and can directly participate in physiological processes. Tyrosine has multiple roles in life activities, involving the nervous system, endocrine system, immune system, and metabolic processes. Here are a few important roles of tyrosine in life activities:

[0003] Neurotransmitter synthesis: Tyrosine is a precursor substance for several important neurotransmitters, including dopamine, adrenaline, and norepinephrine. These neurotransmitters are involved in the normal functioning of the central and peripheral nervous systems, such as regulating mood, controlling movement, and regulating visceral activity.

[0004] Melanin synthesis: Tyrosine is a key substance for melanin synthesis. Melanin is a pigment in the skin, hair, and eyes that plays a decisive role in the color of the eyes and skin, and can absorb ultraviolet rays, protecting the body from ultraviolet damage.

[0005] Immune regulation: Tyrosine can affect and regulate the immune system. It is an energy source for immune cells and can enhance the activity and function of immune cells to resist the invasion of pathogens.

[0006] Metabolism and growth and development: Tyrosine is involved in protein metabolism in the body and is an important component of muscle protein synthesis. It plays an important role in maintaining normal growth and tissue repair of the body.

[0007] Therefore, there is an urgent need in the fields of medical diagnosis, physiological research, and nutrition science to develop detection and analysis techniques for L-Tyr that are simple, sensitive, and efficient.

[0008] Currently, there are many methods for detecting L-Tyr, including traditional methods such as high-performance liquid chromatography, gas chromatography, and capillary electrophoresis. However, these methods have the disadvantages of expensive equipment, complicated operation, and low sensitivity. In contrast, fluorescence sensing technology has the advantages of high selectivity, high sensitivity, low detection limit, and simple operation. Therefore, using a fluorescent probe to specifically recognize L-Tyr is a method worth promoting. SUMMARY

[0009] The application aims to provide a preparation method and application of a fluorescent probe capable of specifically recognizing high-concentration L-Tyr in an aqueous phase, and aims to solve the technical problem of obtaining a fluorescent probe structure capable of specifically recognizing high-concentration L-Tyr in an aqueous phase through molecular design, so as to realize specific recognition of L-Tyr in the 20 kinds of amino acids in the human body, and to have the advantages of specific selectivity, high sensitivity, wide detection concentration range and the like.

[0010] The above technical purpose of the application is achieved by the following technical scheme:

[0011] A fluorescent probe for detecting L-tyrosine (L-Tyr), and the specific chemical structure is as follows:

[0012]

[0013] The application further provides a preparation method of the fluorescent probe for detecting L-tyrosine (L-Tyr), and the method comprises the following steps:

[0014] S1, 4-chloro-1,8-naphthalene dianhydride is weighed and dissolved in a proper amount of 1,4-dioxane, fully stirred and dissolved, n-butylamine is weighed and dissolved in a proper amount of tetrahydrofuran, fully stirred and dissolved, the two solutions are fully mixed, heated and refluxed at 120 DEG C for 24 hours, after the reaction is completed, the temperature is restored to room temperature, the reaction solvent is removed by rotary evaporation, extracted with ethyl acetate, the organic phase is combined and dried with anhydrous sodium sulfate, and rotary evaporation is performed to obtain a yellow solid crude product, which is purified by column chromatography to obtain a yellow intermediate (R)-2;

[0015] S2, the intermediate (R)-2 and N-hydroxy phthalimide are weighed and dissolved in a proper amount of dimethyl sulfoxide according to a molar ratio of 1:1.1, the raw materials are fully stirred and dissolved, a proper amount of anhydrous K2CO3 is added, heated and refluxed at 80 DEG C for 6 hours, after the reaction is completed, the temperature is restored to room temperature, a proper amount of ultrapure water is added to the reaction mixture, hydrochloric acid is used to adjust the pH value to 3, and a yellow intermediate (R)-3 is obtained by filtration;

[0016] S3, the intermediate (R)-3 is weighed and dissolved in a proper amount of DMF and acetone, 2-bromoethanol and anhydrous K2CO3 are added, heated and refluxed at 65 DEG C for 4 hours, after the reaction is completed, the temperature is restored to room temperature, the reaction solvent is removed by rotary evaporation, extracted with dichloromethane, the organic phases are combined and dried with anhydrous sodium sulfate, rotary evaporation is performed to obtain a solid crude product, which is purified by column chromatography to obtain a yellow intermediate (R)-4;

[0017] S4, the intermediate (R)-4 was weighed into an appropriate amount of pyridine, p-toluenesulfonyl chloride was added, stirred at room temperature for 4 h, then an appropriate amount of 5% mass fraction hydrochloric acid was added, and stirring was continued for 1 h. Ethyl acetate was used for extraction, the organic phases were combined, anhydrous sodium sulfate was used for drying, and rotary evaporation was performed to obtain a yellow solid crude product. Column chromatography was used for purification to obtain a yellow intermediate (R)-5.

[0018] S5, the S-naphthol was weighed into an appropriate amount of super-dry dichloromethane, N,N-diisopropyl ethylamine was slowly added at 0°C, and the reaction was allowed to proceed for 3 h. Bromomethyl methyl ether was then slowly added, and the reaction was allowed to proceed for 1 h. Pure water was used to quench the reaction, dichloromethane was used for extraction, the organic phases were combined, anhydrous sodium sulfate was used for drying, and rotary evaporation was performed to obtain a yellow solid crude product. Column chromatography was used for purification, and vacuum drying was performed to obtain a white intermediate (R)-7.

[0019] S6, the intermediate (R)-7 was weighed into an appropriate amount of super-dry tetrahydrofuran, the solution was cooled to -78°C, and n-butyl lithium was added dropwise. The temperature was raised to 0°C, and the reaction was allowed to proceed for 2 h. The solution turned black, super-dry N-N dimethylformamide was added dropwise, the reaction was allowed to proceed for 3 h, and a saturated ammonium chloride solution was added under an ice water bath to quench the reaction. Ethyl acetate was used for extraction, the organic phases were combined, anhydrous sodium sulfate was used for drying, and rotary evaporation was performed to obtain a yellow solid crude product. Column chromatography was used for purification, and vacuum drying was performed to obtain an intermediate (R)-8.

[0020] S7, the intermediate (R)-5 was weighed into a two-necked flask, vacuum was applied, and nitrogen was circulated three times. An appropriate amount of anhydrous acetonitrile was added to dissolve the raw material, and then (R)-8 was added. After 15 min, an appropriate amount of anhydrous K2CO 3， The reaction was allowed to proceed under a nitrogen atmosphere at 80°C for 48 h. After the reaction was completed, the temperature was returned to room temperature, the reaction solvent was removed by rotary evaporation, dichloromethane was used for extraction, the organic phases were combined, anhydrous sodium sulfate was used for drying, and rotary evaporation was performed to obtain a yellow solid crude product. Column chromatography was used for purification to obtain a yellow intermediate (R)-9.

[0021] S8, the intermediate (R)-9 was weighed, an appropriate amount of anhydrous ethanol and dichloromethane was added, an appropriate amount of concentrated hydrochloric acid was added, and stirring was continued for 4 h. Sodium bicarbonate was added to adjust the pH to neutral, dichloromethane was used for extraction, the organic phases were combined, anhydrous sodium sulfate was used for drying, and rotary evaporation was performed to obtain a fluorescent probe powder (R)-10.

[0022] Further preferably, in step S1, the elution phase volume ratio of column chromatography purification is petroleum ether: ethyl acetate = 20:1, and the molar ratio of (1): n-butylamine is 2.6:1.

[0023] Further preferably, in step S3, the elution phase volume ratio of column chromatography purification is petroleum ether: ethyl acetate = 5:1, and the molar ratio of (R)-3: 2-bromoethanol is 2.6:1.

[0024] Further preferably, in step S4, the elution phase volume ratio of the column chromatography purification is petroleum ether: ethyl acetate = 5:1, and the molar ratio of (R)-4: p-toluenesulfonyl chloride is 2.6:1.

[0025] Further preferably, in step S5, the elution phase volume ratio of the column chromatography purification is petroleum ether: ethyl acetate = 40:1, and the molar ratio of S- binaphthol: N,N-diisopropyl ethylamine: bromomethyl methyl ether is 1:2.2:1.1.

[0026] Further preferably, in step S6, the elution phase volume ratio of the column chromatography purification is petroleum ether: ethyl acetate = 10:1, and the molar ratio of (R)-7: n-butyllithium: N-N dimethyl formamide is 1:4:1.5.

[0027] Further preferably, in step S7, the elution phase volume ratio of the column chromatography purification is petroleum ether: ethyl acetate = 6:1, and the molar ratio of (R)-5: (R)-8 is 1:1.5.

[0028] Further preferably, the reactions in steps S5-S7 are carried out under nitrogen protection.

[0029] The application provides a preparation method of a fluorescent probe for detecting L-tyrosine (L-Tyr), comprising the following steps: adding a tetrabutylammonium hydroxide solution with a proper concentration into tyrosine, generating a tyrosine TBA salt after 2 hours of reaction, dissolving the fluorescent probe in chromatographic grade dimethyl sulfoxide to prepare a probe solution, then adding the probe solution, an amino acid TBA salt solution and a certain volume of ultrapure water into a centrifuge tube in sequence, diluting to constant volume after a period of reaction, and performing fluorescence testing, wherein the molar ratio of the fluorescent probe to the amino acid TBA salt is 1:1-100.

[0030] Compared with the prior art, the application has the following beneficial effects:

[0031] Firstly, the fluorescent probe provided by the application is simple to prepare and low in cost.

[0032] Secondly, the fluorescent probe can recognize tyrosine from a plurality of amino acids in ultrapure water.

[0033] Thirdly, the detection method provided by the application is simple to operate, uses a solvent which is low in price and convenient to handle. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 The synthesis route of the fluorescent probe (R)-10 of the application;

[0035] Figure 2 (a) the nuclear magnetic resonance hydrogen spectrum of (R)-10; (b) the nuclear magnetic resonance carbon spectrum of (R)-10.

[0036] Figure 3 (a) fluorescence spectra of (R)-10 recognizing 20 kinds of amino acids; (b) fluorescence enhancement contrast of (R,(R)-7 recognizing 20 kinds of amino acids at 542 nm;

[0037] Figure 4 (a) fluorescence spectra of the fluorescent probe (R)-10 of the present application and L-tyrosine at different times; (b) fluorescence intensity of (R)-10 at 542 nm at different times after reacting with L-tyrosine;

[0038] Figure 5 (a) fluorescence spectra of (R)-10 reacting with L-tyrosine of different concentrations; (b) fluorescence intensity of (R)-10 at 542 nm after reacting with L-tyrosine of different concentrations;

[0039] Figure 6 NMR titration experiment of (R)-10 and L-tyrosine;

[0040] Figure 7 (a) (R)-10; (b) mass spectrum of (R)-10 and L-tyrosine. DETAILED DESCRIPTION

[0041] In order for those skilled in the art to better understand the technical solutions of the present application, the present application will be further described in detail below in combination with examples. Those skilled in the art will understand that the following examples are only used to illustrate the present application, and should not be regarded as limiting the scope of the present application.

[0042] Example 1, Reference Figure 1 A preparation method of a fluorescent probe for detecting L-tyrosine (L-Tyr):

[0043] S1, add a solution of (1) (5 g, 21.0 mmol) dissolved in 1,4-dioxane to a turbid solution of n-butylamine (0.8 ml, 8 mmol) dispersed in tetrahydrofuran, (1) is 4-chloro-1,8-naphthalic anhydride. Stir well, then heat to reflux at 120°C for 24 h, the solution turns into a light yellow turbid solution. Monitor the reaction completion by TLC plate. The reacted turbid solution is rotary evaporated to remove the solvent, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated by vacuum evaporation. The crude product is separated by thin layer chromatography in a solution of petroleum ether and ethyl acetate to obtain the yellow solid intermediate (R)-2.

[0044] S2, Weigh intermediate (R)-2 (2 g, 6.9 mmol) and N-hydroxyphthalimide (1.2 g, 7.4 mmol) into 100 mL dimethyl sulfoxide, fully stir the raw material to dissolve, the solution is light yellow. Add anhydrous K2CO3 (3 g, 21.7 mmol), the solution becomes dark brown, heat to reflux at 80°C for 6 h, monitor the reaction completion by TLC plate. After the reaction is completed, solid precipitates at the bottom, cool to room temperature, add 200 mL ultrapure water to the reaction mixture, adjust the pH to 3 with hydrochloric acid, and filter to obtain yellow intermediate (R)-3.

[0045] S3, Weigh intermediate (R)-3 (0.8 g, 3 mmol) into N,N-dimethylformamide (2 mL, 26 mmol) and 20 mL acetone, add 2-bromoethanol (2.2 mL, 30 mmol), fully stir to mix, add anhydrous K2CO3 (621 mg, 6 mmol), heat to reflux at 65°C for 4 h, cool to room temperature after the reaction is completed, rotary evaporation to remove the reaction solvent, extract with ethyl acetate, dry with anhydrous sodium sulfate, and vacuum evaporate to concentrate. The crude product is separated by thin layer chromatography in a solution of petroleum ether and ethyl acetate to obtain yellow powder (R)-4.

[0046] S4, Weigh intermediate (R)-4 (768 mg, 2.4 mmol) into 5 mL pyridine, slowly add p-toluenesulfonyl chloride (1.42 g, 7.4 mmol), stir at room temperature for 4 h, add 50 mL 5% hydrochloric acid, continue to stir for 1 h, extract with ethyl acetate after the reaction is completed, combine the organic phases, dry with anhydrous sodium sulfate, and rotary evaporation to obtain light yellow solid crude product, which is separated by thin layer chromatography in a solution of petroleum ether and ethyl acetate to obtain yellow intermediate (R)-5.

[0047] S5, Dissolve (R)-1 (10.0 g, 34.9 mmol) in 100 mL ultrapure dichloromethane under a nitrogen atmosphere. Cool the reaction solution to 0°C, slowly add N,N-diisopropylethylamine (10.3 mL, 76.8 mmol), and react for 3 h, and then the solution is clear and transparent. Slowly add bromomethyl methyl ether (3.13 mL, 38.4 mmol), react for 1 h, and then the solution turns slightly yellow. Monitor the reaction completion by TLC plate. Quench the reaction by adding ultrapure water, extract with dichloromethane, combine the organic phases, dry with anhydrous sodium sulfate, and vacuum evaporate to concentrate. The crude product is separated by thin layer chromatography in a solution of petroleum ether and ethyl acetate to obtain white powder (R)-7.

[0048] S6, (R)-7 (6.00 g, 18.3 mmol) was dissolved in 100 mL of tetrahydrofuran under nitrogen atmosphere. The solution was cooled to -78 °C, and n-butyllithium (1.6 M hexane solution, 45.7 mL, 73.2 mmol) was added dropwise slowly. The solution was warmed to 0 °C for 2 h, and turned black. Dry N-N dimethylformamide (2.11 mL, 27.4 mmol) was added gradually, and the solution turned orange. The reaction was monitored by TLC plate, and the reaction was completed. The completed reaction solution was poured into a cold saturated ammonium chloride solution, and the solution turned yellow. The solution was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated by evaporation under vacuum. The crude product was separated by thin layer chromatography in a solution of petroleum ether and ethyl acetate to obtain a yellow powder (R)-8.

[0049] S7, (R)-5 (0.48 g, 1.1 mmol) was dissolved in 30 mL of anhydrous acetonitrile under nitrogen atmosphere. (R)-8 (0.57 mg, 1.1.67 mmol) was added, and after 15 min, anhydrous K2CO3 (570 mg, 4.28 mmol) was added. The solution was heated to reflux at 80 °C for 48 h. After the reaction was completed, the solution was cooled to room temperature, and the reaction solvent was removed by rotary evaporation. The solution was extracted with dichloromethane, and the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated by rotary evaporation to obtain a yellow solid crude product. The crude product was separated by thin layer chromatography in a solution of petroleum ether and ethyl acetate to obtain a yellow intermediate (R)-9.

[0050] S8, The intermediate (R)-9 (2 g, 3 mmol) was weighed, and 20 mL of anhydrous ethanol and 20 mL of dichloromethane were added. 1 mL of concentrated hydrochloric acid was added, and the solution was stirred for 4 h. Sodium bicarbonate was added to adjust the pH to 7, and the solution was extracted with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated by rotary evaporation to obtain a fluorescent probe powder (R)-10.

[0051] Example 2, a detection method of a fluorescent probe for detecting L-tyrosine (L-Tyr):

[0052] The probe (R)-10 was dissolved in an appropriate amount of chromatographic grade dimethyl sulfoxide to prepare a 1.6 mM probe solution, and the amino acid was dissolved in a tetrabutylammonium hydroxide solution to react for 2 h to prepare a 1-100 eq amino acid TBA salt solution. Subsequently, 50 ul of the probe solution and 50 ul of the amino acid TBA salt solution were sequentially added to a centrifuge tube, and the molar ratio of the fluorescent probe to the amino acid TBA salt was 1:1-100. After dilution with 3.9 mL of dimethyl sulfoxide, the fluorescence test was performed after 3 h, and the fluorescence test parameters were as follows: excitation slit, 5 nm; emission slit, 5 nm; and excitation wavelength, 430 nm.

[0053] Reference Figure 2, (a) is the nuclear magnetic resonance hydrogen spectrum of the fluorescent probe (R)-10 in deuterated chloroform, (b) is the nuclear magnetic resonance carbon spectrum of the fluorescent probe (R)-10 in deuterated chloroform;

[0054] Referring to Figure 3 , the probe (R)-10 itself has no obvious fluorescence in dimethyl sulfoxide, and when the probe (R)-10 reacts with 20 kinds of different amino acids with 100 eq, only L-Tyr shows obvious fluorescence enhancement at λ=542 nm; when (R)-10 interacts with other 19 kinds of amino acids, the fluorescence intensity is significantly reduced, and some even have no fluorescence response;

[0055] Referring to Figure 4 , when the fluorescent probe (R)-10 reacts with L-Tyr at different times, the reaction time interval is 0-300 min, and it can be seen that when the reaction time is 180 min, the fluorescence intensity reaches a plateau;

[0056] Referring to Figure 5 , when the fluorescent probe (R)-10 reacts with L-Tyr at different equivalent concentrations, the measured equivalent interval is 0-100 eq, and it can be seen that when L-Tyr is 100 eq, the fluorescence intensity reaches a maximum.

[0057] Referring to Figure 6 , it is the nuclear magnetic mechanism diagram of the fluorescent probe (R)-10 and different equivalent L-Tyr, and it can be seen that the aldehyde group peak between the displacement 9.5-10.5 disappears, which proves that the probe reacts completely with tyrosine, and a new peak is generated between the displacement 6-7, which proves that the probe reacts with tyrosine to form a new structure.

[0058] Referring to Figure 7 , (a) is the mass spectrum result of the fluorescent probe (R)-10, the molecular weight of the predicted probe (R)-10 is 609.22, m / z=608.22 [m-1] is found in the mass spectrum, which is consistent with the predicted result, confirming that the obtained product is the target compound probe (R)-10, (b) is the mass spectrum result after the reaction of the probe (R)-10 and L-Tyr, the molecular weight of the generated product is predicted to be 772.30, m / z=773.30 [m+1] is found in the mass spectrum, which is consistent with the predicted result, confirming that the generated product is the target compound. The fluorescent probe of the present application can recognize L-configuration tyrosine in ultrapure water.

[0059] The detection method provided by the present application is simple to operate, and the solvent is low in price and convenient to post-process.

[0060] The embodiments are only used for explaining the present application, and are not used for limiting the present application, and the person skilled in the art can make the modification of the embodiments without the creative contribution according to the need after reading the description, and as long as the modification is within the scope of the claims of the present application, it is protected by the patent law.

Claims

1. A fluorescent probe for detecting L-tyrosine (L-Tyr), characterized by, The specific chemical structure is shown as follows: 。 2. The method according to claim 1, wherein the method for preparing a fluorescent probe for detecting L-tyrosine (L-Tyr) is characterized by, The method comprises the following steps: S1, 4-chloro-1,8-naphthalene dianhydride is weighed and dissolved in 1,4-dioxane, n-butylamine is weighed and dissolved in tetrahydrofuran, the two solutions are fully mixed, and the mixture is heated to reflux at 120 DEG C for 24 h; after the reaction is completed, the temperature is restored to room temperature, and the reaction solvent is removed by rotary evaporation; the obtained product is extracted with ethyl acetate, the organic phases are combined, dried over anhydrous sodium sulfate, and then rotary evaporation is performed to obtain a yellow solid crude product; column chromatography is performed to purify the product, and a yellow intermediate (R)-2, 2-butyl-6-chloro-1H-benzo[de] isoquinoline-1,3(2H)-dione is obtained; S2, the intermediate (R)-2 and N-hydroxyphthalimide are weighed and dissolved in dimethyl sulfoxide according to a molar ratio of 1:1.1, the raw materials are fully stirred and dissolved, and then anhydrous K2CO3 is added; the mixture is heated to reflux at 80 DEG C for 6 h; after the reaction is completed, the temperature is restored to room temperature, and then ultrapure water is added to the reaction mixture; the pH value is adjusted to 3 by using hydrochloric acid; and then filtration is performed to obtain a yellow intermediate (R)-3, 2-butyl-6-hydroxy-1H-benzo[de] isoquinoline-1,3(2H)-dione; S3, the intermediate (R)-3 is dissolved in DMF and acetone, 2-bromoethanol and anhydrous K2CO3 are added, and the mixture is heated to reflux at 65 DEG C for 4 h; after the reaction is completed, the temperature is restored to room temperature, and then the reaction solvent is removed by rotary evaporation; the obtained product is extracted with dichloromethane, the organic phases are combined, dried over anhydrous sodium sulfate, and then rotary evaporation is performed to obtain a solid crude product; column chromatography is performed to purify the product, and a yellow intermediate (R)-4, 2-butyl-6-(2-hydroxyethoxy)-1H-benzo[de] isoquinoline-1,3(2H)-dione, is obtained; S4, the intermediate (R)-4 is dissolved in pyridine, and p-toluenesulfonyl chloride is added; the mixture is stirred at room temperature for 4 h, and then 5% hydrochloric acid is added; the mixture is continuously stirred for 1 h, extracted with ethyl acetate, and then the organic phases are combined and dried over anhydrous sodium sulfate; rotary evaporation is performed to obtain a yellow solid crude product; column chromatography is performed to purify the product, and a yellow intermediate (R)-5, 2-((2-butyl-1,3-dioxo-2,3-dihydro-1H-benzo[de] isoquinoline-6-yl)oxy)ethyl 4-methyl phenyl sulfonate, is obtained; S5, R-binol is dissolved in ultradry dichloromethane, N,N-diisopropyl ethylamine is slowly added at 0 DEG C, the mixture is reacted for 3 h, and then bromomethyl methyl ether is slowly added; the mixture is reacted for 1 h, quenched by adding ultrapure water, extracted with dichloromethane, and then the organic phases are combined and dried over anhydrous sodium sulfate; rotary evaporation is performed to obtain a yellow solid crude product; column chromatography is performed to purify the product, and vacuum drying is performed to obtain a white intermediate (R)-7, 2'-(methoxymethoxy)-[1,1'-binaphthalen]-2-ol. S6, the intermediate (R)-7 was weighed and dissolved in super dry tetrahydrofuran, the solution was cooled to -78 ℃, n-butyllithium was added dropwise, and the temperature was raised to 0 ℃ for 2 h, the solution turned black, super dry N,N-dimethylformamide was added dropwise, and the reaction was carried out for 3 h, saturated ammonium chloride solution was added under ice water bath condition to quench the reaction, ethyl acetate was extracted, the organic phase was combined, anhydrous sodium sulfate was dried, and rotary evaporation was carried out to obtain yellow solid crude product, column chromatography was used for purification, and vacuum drying was carried out to obtain the intermediate (R)-8, 2'-hydroxy-2-(methoxymethoxy)-[1,1'-binaphthalen]-3-carbaldehyde; S7, the intermediate (R)-5 in two mouth bottles, vacuum nitrogen circulation three times, the addition of anhydrous acetonitrile to dissolve the raw material, then add intermediate (R)-8, 15 min, the addition of anhydrous K2CO 3， under nitrogen atmosphere, heating at 80℃ temperature conditions for 48h, the reaction was completed after recovery to room temperature, rotary evaporation to remove the reaction solvent, dichloromethane extraction, combined organic phase, anhydrous sodium sulfate drying, rotary evaporation to obtain light yellow solid crude product, column chromatography purification, obtained yellow intermediate (R)-9 as 2'-(2-((2-butyl-1,3-diketone-2,3-dihydro-1H-benzo[de] isoquinoline-6-yl)oxy)ethoxy)-2-(methoxymethoxy)-[1,1'-binaphthalene]-3-carboxaldehyde; S8, the intermediate (R)-9 was weighed, anhydrous ethanol and dichloromethane were added, concentrated hydrochloric acid was added, and stirring was carried out for 4 h, sodium bicarbonate was added to adjust the pH to neutral, dichloromethane was used for extraction, the organic phase was combined, anhydrous sodium sulfate was dried, and rotary evaporation was carried out to obtain fluorescent probe powder (R)-10, 2'-(2-((2-butyl-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)oxy)ethoxy)-2-hydroxy-[1,1'-binaphthalen]-3-carbaldehyde.

3. The method for preparing a fluorescent probe for detecting L-tyrosine (L-Tyr) according to claim 2, characterized in that: In step S1, the elution phase of column chromatography purification was petroleum ether: ethyl acetate = 20:1 in volume ratio, and the molar ratio of (R)-1 to n-butylamine was 2.6:

1. 4.The method for preparing a fluorescent probe for detecting L-tyrosine (L-Tyr) according to claim 2, characterized in that: In step S3, the elution phase of column chromatography purification was petroleum ether: ethyl acetate = 5:1 in volume ratio, and the molar ratio of (R)-3 to 2-bromoethanol was 2.6:

1. 5.The method for preparing a fluorescent probe for detecting L-tyrosine (L-Tyr) according to claim 2, characterized in that: In step S4, the elution phase of column chromatography purification was petroleum ether: ethyl acetate = 5:1 in volume ratio, and the molar ratio of (R)-4 to p-toluenesulfonyl chloride was 2.6:

1. 6.The method for preparing a fluorescent probe for detecting L-tyrosine (L-Tyr) according to claim 2, characterized in that: In step S5, the elution phase of column chromatography purification was petroleum ether: ethyl acetate = 40:1 in volume ratio, and the molar ratio of R-binafol to N,N-diisopropylethylamine to bromomethyl methyl ether was 1:2.2:1.

1.

7. The method for preparing a fluorescent probe for detecting L-tyrosine (L-Tyr) according to claim 2, characterized in that: In step S6, the elution phase of column chromatography purification was petroleum ether: ethyl acetate = 10:1 in volume ratio, and the molar ratio of (R)-7 to n-butyllithium to N,N-dimethylformamide was 1:4:1.

5. 8.The method for preparing a fluorescent probe for detecting L-tyrosine (L-Tyr) according to claim 2, characterized in that: In step S7, the elution phase of column chromatography purification was petroleum ether: ethyl acetate = 6:1 in volume ratio, and the molar ratio of (R)-5 to (R)-8 was 1:1.

5.

9. The method for preparing a fluorescent probe for detecting L-tyrosine (L-Tyr) according to any one of claims 2-8, characterized in that: The reactions of steps S5-S7 were all carried out under nitrogen protection.

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