Preparation method and detection method of a specific selective recognition tryptophan fluorescent probe

Abstract

This invention discloses a method for preparing and detecting a fluorescent probe that specifically and selectively recognizes tryptophan, belonging to the field of fluorescence detection technology. (R)-1 is dissolved in DCM, and reacted after adding DIPEA; bromomethyl methyl ether is added and reacted again; HCl is added and extracted with ethyl acetate, and the organic phases are combined; impurity solvents are removed to obtain a crude product, which is then purified to obtain (R)-5. (R)-5 is fully dissolved, and n-butyllithium is added dropwise to the cooled solution; DMF is added dropwise after cooling and extraction is performed, the organic phases are combined, and the impurity solvents are removed by drying to obtain a purified crude product; product (R)-4 is obtained; (R)-4 is dissolved in ultra-dry acetonitrile; DIPEA is added dropwise at a lower temperature and reacted for 2 hours; acryloyl chloride is added dropwise at 0℃ and reacted; the organic phases are extracted and combined; impurity solvents are removed and the crude product is purified to obtain (R)-1. This invention, through the design of the recognition group, realizes the transformation of the probe from recognizing L-Arg to recognizing L-Trp, providing a new approach for the specific and selective fluorescent recognition of tryptophan, and also providing new ideas for subsequent research on specific fluorescent recognition of tryptophan.

Description

Technical Field

[0001] This invention belongs to the field of fluorescence detection technology, specifically relating to a method for preparing and detecting a fluorescent probe that specifically and selectively recognizes tryptophan. Background Technology

[0002] In the human body, tryptophan is one of the essential amino acids and a basic building block of proteins. It is also a precursor to serotonin, an important neurotransmitter, playing a crucial role in maintaining the normal function of the nervous system. Tryptophan has significant effects on regulating nerve rhythms and improving sleep. In animals, it promotes the function of riboflavin and contributes to the synthesis of niacin and heme in the process of plasma protein turnover. During pregnancy, tryptophan significantly increases antibodies in the fetus and promotes lactation in lactating cows and sows. A deficiency of tryptophan in humans can lead to growth retardation, weight loss, and reduced fat accumulation. In plants, tryptophan is an important precursor in the biosynthesis of auxin, playing a key regulatory role in plant growth and development. In summary, tryptophan plays an indispensable role in both human health and plant growth. Therefore, it has wide applications in nutritional supplements, specialty milk powders, and pharmaceuticals. Thus, tryptophan recognition technology is particularly important. Through specific selective recognition, we can accurately distinguish this amino acid, ensuring its safety and effectiveness in various applications. This is not only crucial for the development of the chemical industry, but also provides new perspectives and tools for research in the life sciences. Fluorescence detection technology, with its advantages of high sensitivity, high selectivity, and high-throughput analysis, was chosen as the detection method. In this work, we designed and synthesized a novel fluorescent probe (R)-1 with multiple reactive sites, including aldehyde, hydroxyl, and acrylate groups, based on the BINOL backbone, and successfully achieved specific recognition of tryptophan in methanol.

[0003] The high structural similarity among various amino acids makes specific amino acid recognition extremely challenging. Furthermore, the interaction between different configurations of the same amino acid can reduce the probe's specific recognition ability for a particular amino acid. More notably, most current BINOL-based amino acid fluorescent probes contain aldehyde groups in their recognition groups, which can lead to a decrease in probe specificity as amino acid concentration increases. Simultaneously, most existing BINOL-based amino acid fluorescent probes exhibit high enantioselectivity, responding to a wide range of amino acids, but rarely specifically recognizing a particular amino acid. Therefore, achieving a specific and selective fluorescent response of BINOL to a specific amino acid remains a highly challenging task. In previous research, Zeng et al. achieved the specific and selective recognition of arginine in methanol using acrylate-containing BINOL fluorescent probes. However, current research results indicate that such probes do not show good recognition performance for other L-amino acids, and BINOL fluorescent probes based on acrylate groups and their derivatives still face many challenges. Therefore, in this work, we retained the original BINOL backbone and replaced the original recognition group with an acrylate group with a smaller spatial structure. We designed and synthesized a novel fluorescent probe (R)-1 with multiple reaction sites of aldehyde, hydroxyl and acrylate groups based on BINOL backbone, and successfully achieved specific recognition of tryptophan in methanol. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a method for preparing and detecting a fluorescent probe that specifically and selectively recognizes tryptophan.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] This invention provides a method for preparing a fluorescent probe that specifically and selectively recognizes tryptophan, comprising the following steps:

[0007] S1. Weigh (R)-6, i.e. (R)-BINOL, dissolve it in ultra-dry DCM, cool the solution to 0℃, add DIPEA dropwise and react for 3h; add bromomethyl methyl ether dropwise and continue the reaction for 1h.

[0008] S2. Then, HCl was added at 0°C to quench the reaction; then, ethyl acetate was used for multiple extractions, and the resulting organic phases were combined; the mixture was dried with anhydrous Na2SO4 and filtered to remove impurities.

[0009] S3. The solvent was removed by vacuum to obtain the crude product; the crude product was further purified by column chromatography to obtain the white solid product (R)-5, namely 2'-(methoxymethoxy)-[1,1'-binaphthyl]-2-ol;

[0010] S4. Dissolve (R)-5 completely in ultra-dry THF and cool the solution to -78°C; then add n-butyllithium dropwise, restore to room temperature and continue the reaction for 2 hours; cool to 0°C again, add DMF dropwise, and then restore to room temperature and react for 1 hour.

[0011] S5. Cool to 0℃ again, add saturated NH4Cl to quench the reaction, and extract with ethyl acetate multiple times. Then combine the organic phases, dry with anhydrous Na2SO4, and filter to remove impurities.

[0012] S6. The solvent was removed by vacuum to obtain the crude product. The crude product was further purified by column chromatography to obtain the yellow oily product (R)-4, namely 2'-hydroxy-2-(methoxymethoxy)-[1,1'-binaphthyl]-3-carboxaldehyde.

[0013] S7. Add (R)-4 to a 250ml round-bottom flask, evacuate and purge with nitrogen three times, add ultra-dry acetonitrile to dissolve completely; cool to 0℃ and add DIPEA dropwise, then restore to room temperature and react for 2h, cool to 0℃ again and add acryloyl chloride dropwise, and after the addition is complete, restore to room temperature and react for 3h.

[0014] S8. Add Ce(SO4)2·4H2O and reflux for 8 hours, then extract multiple times with ethyl acetate and combine the resulting organic phases.

[0015] S9. The product was dried with anhydrous Na2SO4 and filtered to remove impurities. The solvent was removed by vacuum to obtain the crude product. The crude product was further purified by column chromatography to obtain the yellow solid product (R)-1, namely 3'-formyl-2'-hydroxy-[1,1'-binaphthyl]-2-yl ester of acrylate.

[0016] Further, in step S3, the eluent used in column chromatography is a mixture of petroleum ether and ethyl acetate in a ratio of 40:1; in step S6, the eluent used in column chromatography is a mixture of petroleum ether and ethyl acetate in a ratio of 10:1; and in step S9, the eluent used in column chromatography is a mixture of petroleum ether and ethyl acetate in a ratio of 20:1.

[0017] Furthermore, it also includes a detection method for a fluorescent probe that specifically and selectively recognizes tryptophan, using solvents DMSO, ACN, H2O, THF, MeOH, and DMF to test the effect of (R)-1 on the recognition of tryptophan.

[0018] Furthermore, using MeOH as a solvent, the fluorescence response of (R)-1 to glycine and 18 other free amino acids was studied.

[0019] Furthermore, zinc ion detection (Zn) was added to the detection of L-Trp. 2+The effect of concentration on the test results.

[0020] Furthermore, (R)-1 was used to detect different concentrations of L-Trp.

[0021] The beneficial effects of this invention are as follows:

[0022] 1. In order to expand the detection field of BINOL probes with acrylate groups, this invention designs and synthesizes a novel fluorescent probe (R)-1 with multiple reaction sites of aldehyde, hydroxyl and acrylate groups with BINOL as the backbone, and successfully achieves specific recognition of tryptophan in methanol.

[0023] 2. The results of this invention show that after 4 hours of reaction, (R)-1 can show a significant fluorescence response to a specific concentration of tryptophan in methanol solution, while the fluorescence response to other L-amino acids is almost negligible.

[0024] 3. The mechanism analysis of this invention suggests that in methanol solution, probe (R)-1 and L-Trp interact under intermolecular forces, and in Zn 2+ Stable compounds were formed under coordination, thus achieving fluorescent recognition. Other amino acids, due to configurational mismatch, are difficult to form stable compounds. By designing the recognition group, the probe was transformed from recognizing L-Arg to recognizing L-Trp, providing a new approach for the specific and selective fluorescent recognition of tryptophan and offering new ideas for subsequent research on specific fluorescent recognition of tryptophan.

[0025] Other advantages, objectives, and features of the invention will be set forth in the following description and will be apparent to those skilled in the art in some respects, or may be learned by practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0026] To make the purpose, technical solution, and beneficial effects of the invention clearer, the following figures are provided for illustration:

[0027] Figure 1 This is a synthetic route diagram of the present invention (R)-1;

[0028] Figure 2 The proton NMR spectrum (400 MHz) of the probe (R)-5 of this invention dissolved in CDCl3;

[0029] Figure 3 The carbon NMR spectrum (101 MHz) of the probe (R)-5 of this invention dissolved in CDCl3;

[0030] Figure 4The proton NMR spectrum (400 MHz) of (R)-4 dissolved in CDCl3 in this invention;

[0031] Figure 5 The carbon NMR spectrum (101 MHz) of (R)-4 dissolved in CDCl3 according to the present invention;

[0032] Figure 6 The proton NMR spectrum (400 MHz) of probe (R)-1 of this invention using DMSO-d6 as solvent;

[0033] Figure 7 The carbon NMR spectrum (101 MHz) of probe (R)-1 of this invention using DMSO-d6 as solvent;

[0034] Figure 8 shows the fluorescence spectra of probe (R)-1 after reaction with tryptophan in DMSO, ACN, H2O, THF, MeOH and DMF solutions, respectively.

[0035] Figure 9(a) shows the addition of 30 eq L-amino acids and 3 eq Zn to (0.8 mM)(R)-1 in MeOH according to the present invention. 2+ Fluorescence response after 4 hours of reaction;

[0036] Figure 9(b) shows the addition of 30 eq L-amino acids and 3 eq Zn to (0.8 mM)(R)-1 in MeOH according to the present invention. 2+ Fluorescence spectrum after 4 hours of reaction;

[0037] Figure 9(c) shows the addition of 3 eq Zn to (R)-1 (0.8 mM) in MeOH according to the present invention. 2+ Fluorescence spectrum of reaction with 30 eq L-Trp for 4 h;

[0038] Figure 10(a) shows the addition of 3eq.Zn to (R)-1 according to the present invention. 2+ Fluorescence spectra of the product reacted with 30 eq. L-Trp at different times;

[0039] Figure 10(b) shows the addition of 3eq Zn to (R)-1 according to the present invention. 2+ Fluorescence intensity at 560 nm at different reaction times with 30 eq. L-Trp;

[0040] Figure 10(c) shows the addition of 3eq Zn to (R)-1 according to the present invention. 2+ Fluorescence spectrum after reaction with 30 eq. L-Trp for 4 h;

[0041] Figure 11(a) shows the addition of different equivalents of Zn to (R)-1 according to the present invention. 2+Fluorescence spectrum after reaction with 30 eq. L-Trp for 4 h;

[0042] Figure 11(b) shows the addition of different equivalents of Zn to (R)-1 according to the present invention. 2+ Fluorescence intensity at 560 nm after reaction with 30 eq. L-Trp for 4 h;

[0043] Figure 11(c) shows the addition of 3eq Zn to (R)-1 according to the present invention. 2+ Fluorescence spectrum after reaction with 30 eq. L-Trp for 4 h;

[0044] Figure 12(a) shows the addition of 3eq Zn to (R)-1 according to the present invention. 2+ Fluorescence spectra of different equivalents of L-Trp reacted for 4 hours;

[0045] Figure 12(b) shows the addition of 3eq Zn to (R)-1 according to the present invention. 2+ Fluorescence intensity at 560 nm after reacting with different equivalents of L-Trp for 4 hours;

[0046] Figure 12(c) shows the addition of 3eq Zn to (R)-1 according to the present invention. 2+ Fluorescence spectrum of reaction with 30 eq. L-Trp for 4 h;

[0047] Figure 13(a) shows the addition of 3 eq Zn to probe (R)-1 (0.01 mM) according to the present invention. 2+ UV absorption spectra of reactions with different equivalents of L-Trp;

[0048] Figure 13(b) shows the addition of 3eq Zn to (R)-1 according to the present invention. 2+ UV absorption spectrum of the reaction with 30 eq. L-Trp;

[0049] Figure 14 This invention relates to the experimental reaction of (R)-1 with L-Trp NMR titration.

[0050] Figure 15 The molecular structure that may be generated after the reaction of (R)-1 and L-Trp in this invention;

[0051] Figure 16(a) shows the high-resolution mass spectrum of (R)-1 of the present invention, and Figure 16(b) shows the high-resolution mass spectrum of (R)-1 of the present invention after reaction with L-Trp. Detailed Implementation

[0052] A method for preparing a fluorescent probe that specifically and selectively recognizes tryptophan:

[0053] In this invention, (R)-BINOL is referred to as (R)-6; (2'-(methoxymethoxy)-[1,1'-binaphthyl]-2-ol) is referred to as (R)-5; 2'-hydroxy-2-(methoxymethoxy)-[1,1'-binaphthyl]-3-carboxaldehyde is referred to as (R)-4; and 3'-formyl-2'-hydroxy-[1,1'-binaphthyl]-2-yl ester of acrylate is referred to as (R)-1.

[0054] Example 1

[0055] S1. Weigh (R)-6 (17.5 mmol, 5 g) and dissolve it completely in 100 mL of ultra-dry DCM. Cool the solution to 0 °C and slowly add DIPEA (38.5 mmol, 6.4 mL). React for 3 h. Then slowly add bromomethyl methyl ether (26.2 mmol, 2.1 mL). React for 1 h.

[0056] S2. The reaction was quenched by adding 2M HCl (50mL) at 0℃, followed by three extractions with 20mL ethyl acetate. The resulting organic phases were combined and dried with anhydrous Na2SO4 and filtered to remove impurities.

[0057] S3. The solvent was removed under reduced pressure to obtain the crude product; the crude product was further purified by column chromatography, using a mixture of petroleum ether and ethyl acetate in a ratio of 40:1 as the eluent; 4.0 g of a white solid product (R)-5 was then obtained, with a yield of 80%. 1 H NMR see Figure 2 , 13 C NMR (see) Figure 3 ;

[0058] S4. Dissolve (R)-5 (12.2 mmol, 4 g) completely in 100 mL of ultra-dry THF. Cool the solution to -78 °C and slowly add n-butyllithium (42.8 mmol, 26.8 mL, 1.6 M dissolved in n-hexane). Allow the solution to return to room temperature and react for 2 h. Cool the solution to 0 °C again and slowly add DMF (18.4 mmol, 1.44 mL). Allow the solution to return to room temperature and react for 1 h.

[0059] S5. Cool to 0℃ again, add 20ml of saturated NH4Cl to quench the reaction, and extract three times with 30ml of ethyl acetate. Then combine the organic phases, dry them with anhydrous Na2SO4, and filter to remove impurities.

[0060] S6. The solvent was removed under reduced pressure to obtain the crude product; the crude product was purified by column chromatography using petroleum ether and ethyl acetate in a 10:1 ratio as eluent, yielding 2.2 g of a yellow oily product (R)-4, with a yield of 55%. 1HNMR Figure 4 , 13 C NMR (see) Figure 5 ;

[0061] S7. Add (R)-4 (1.8 mmol, 0.65 g) to a 250 ml round-bottom flask, evacuate and purge with nitrogen three times, add 60 ml of ultra-dry acetonitrile to dissolve completely; cool to 0 °C, slowly add DIPEA (5.4 mmol, 0.9 ml), after the addition is complete, return to room temperature and react for 2 h, cool to 0 °C again, add acryloyl chloride (5.4 mmol, 0.44 ml), after the addition is complete, return to room temperature and react for 3 h;

[0062] S8. Add Ce(SO4)2·4H2O (1.4mmol, 566mg) and reflux for 8h. Extract three times with 20mL of ethyl acetate and combine the resulting organic phases.

[0063] S9. The product was dried over anhydrous Na2SO4 and filtered to remove impurities; the solvent was then removed under reduced pressure to obtain the crude product; the crude product was purified by column chromatography using petroleum ether and ethyl acetate in a 20:1 ratio as eluent; 0.31 g of a yellow solid product (R)-1 was obtained, with a yield of 47%. 1 H NMR see Figure 6 , 13 C NMR (see) Figure 7 .

[0064] In this embodiment, step S3 1 H NMR is represented as follows: 1 H NMR(400MHz,Chloroform-d)δ8.03(d,J=9.1Hz,1H),7.91(d,J=8.6Hz,2H),7.87(d,J=8.1Hz,1H),7.60(d,J=9.1Hz,1H),7.44–7.38(m,1H), 7.36(d,J=8.9Hz,1H),7.31(s,1H),7.30(s,1H),7.23(s,1H),7.21(d,J=8.1Hz,1H),7.08(d,J=8.4Hz,1H),5.13–5.03(m,2H),3.18(s,3H).

[0065] To further demonstrate the superiority of this invention, this application also provides a detection method for a fluorescent probe that specifically and selectively recognizes tryptophan.

[0066] To investigate the effect of different reaction solvents on the recognition of tryptophan by (R)-1, fluorescence tests were performed using different chromatographic solvents (DMSO, ACN, H2O, THF, MeOH, and DMF) under constant conditions. Figures 8(a), (b), (c), (d), (e), and (f) show the fluorescence spectra of probe (R)-1 after reaction with tryptophan in DMSO, ACN, H2O, THF, MeOH, and DMF solutions, respectively. The fluorescence spectra revealed significant changes in fluorescence intensity with the change of reaction solvent. Based on the spectral results, MeOH exhibits higher specificity and fluorescence intensity for (R)-1 recognition of tryptophan compared to other solvents; therefore, MeOH was chosen as the reaction solvent for this study.

[0067] Using MeOH as a solvent, the fluorescence response of (R)-1 to glycine and 18 other free amino acids was studied, and the results are shown in Figure 9(a).

[0068] P1. Add 50 μL of 24.0 mM (30 eq) free amino acids and 50 μL of 2.4 mM (3 eq) Zn sequentially to 50 μL of 0.8 mM (R)-1 solution. 2+ The solution and 250 μL of MeOH were mixed to form a homogeneous solution. The reaction mixture was then allowed to react at room temperature for 240 minutes to allow for complete reaction.

[0069] P2. Dilute the reaction solution to 4 mL with methanol and measure its fluorescence.

[0070] In this process, 50 μL of 0.8 mM standard solution (R)-1 was taken from a centrifuge tube and diluted to 4 mL; the final concentration of probe (R)-1 in the solution was 10 μM. Figures 9(b) and (c) summarize the fluorescence spectral data after (R)-1 interacts with glycine and 18 other free amino acids.

[0071] Figures 9(a), (b), and (c) illustrate the interaction between (R)-1 and different free amino acids and its effect on fluorescence properties by comparing the fluorescence peak intensity at wavelength λ = 560 nm. When (R)-1 interacts with the other 18 L-amino acids and glycine, for most free amino acids, the interaction with (R)-1 results in only a very weak fluorescence enhancement. However, the addition of L-Trp significantly enhances the fluorescence intensity of the solution at wavelength λ = 560 nm. This significant fluorescence enhancement fully demonstrates the excellent chemoselectivity of (R)-1 for L-Trp.

[0072] Under the same solvent system, all other reaction conditions remained unchanged, except for the reaction time of (R)-1 and 30 eq. L-Trp.

[0073] P1. Weigh a certain mass of (R)-1 and dissolve it in chromatographic grade DMSO to prepare a 0.8 mM probe stock solution. Dissolve appropriate amounts of zinc acetate and L-tryptophan separately in ultrapure water to prepare a 2.4 mM Zn solution. 2+ Solution and 24 mM L-tryptophan solution;

[0074] P2. Using a pipette, add 50 μL of probe stock solution, 250 μL of methanol solution, 50 μL of L-tryptophan, and 50 μL of Zn to a centrifuge tube in sequence. 2+ After reacting for a period of time, 3.6 mL of methanol solution was added to bring the volume to 4 mL, and the fluorescence was measured.

[0075] The reaction times were 1 min, 5 min, 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, and 8 h, respectively. The excitation wavelength and emission wavelength were set to 360 nm and 380–700 nm, respectively. The final concentration of (R)-1 in the solution was 10 μM.

[0076] As shown in Figures 10(a), (b), and (c), 3eq.Zn is added through test (R)-1. 2+ The fluorescence intensity changed over time after adding 30 eq. L-Trp. Fluorescence emission increased significantly with increasing reaction time, reaching a high fluorescence intensity within 4 hours, after which the fluorescence intensity remained relatively stable. Considering the high sensitivity of the fluorescence assay, the reaction time for subsequent related tests was set at 4 hours.

[0077] Because zinc ions are added in the detection of L-Trp, Zn also needs to be considered. 2+ The effect of concentration on detection.

[0078] P1. Under the same experimental conditions, prepare Zn solutions of different concentrations. 2+ The solutions were set at concentrations of 0.8 mM (1.0 eq), 1.6 mM (2.0 eq), 2.4 mM (3.0 eq), 3.2 mM (4.0 eq), 4.0 mM (5.0 eq), 4.8 mM (6.0 eq), 5.6 mM (7.0 eq), and 6.4 mM (8.0 eq), respectively.

[0079] P2. Using a pipette, add 50 μL of probe stock solution, 250 μL of methanol solution, 50 μL of L-Trp (30.0 eq), and 50 μL of different equivalents of Zn to a centrifuge tube sequentially. 2+After the solution has reacted fully for 240 min, 3.60 mL of methanol solution was added to bring the volume to 4 mL, and the fluorescence was measured.

[0080] The excitation and emission wavelengths were set to 360 nm and 380–700 nm, respectively, and the final concentration of (R)⁻¹ in the solution was 10 μM. As shown in Figures 11(a), (b), and (c), tests were conducted on the addition of different equivalents of Zn to (R)⁻¹. 2+ The change in fluorescence intensity after 30 eq. of L-Trp. Within the range of 1-3 eq., the fluorescence intensity of L-Trp changes with Zn. 2+ Increased concentration significantly enhanced fluorescence intensity, with a slow increase in the 3-6 eq range, stabilizing at 6-8 eq. Therefore, Zn was used in subsequent related tests. 2+ The concentration was set at 2.4 mM (3.0 eq).

[0081] Further research will be conducted to explore the use of (R)-1 to detect different concentrations of L-Trp.

[0082] P1. Dilute 120 mM L-Trp at a certain ratio to prepare L-Trp solutions with concentrations of 0.8 mM (1 eq), 4.0 mM (5 eq), 8.0 mM (10 eq), 12.0 mM (15 eq), 16 mM (20 eq), 24.0 mM (30 eq), 32.0 mM (40 eq), 40.0 mM (50 eq), 48.0 mM (60 eq), 56.0 mM (70 eq), 64.0 mM (80 eq), 72.0 mM (90 eq), 80.0 mM (100 eq), 96.0 mM (120 eq), and 120.0 mM (150 eq).

[0083] P2. Using a pipette, add 50 μL of probe stock solution, 250 μL of methanol solution, 50 μL of different equivalents of L-Trp, and 50 μL of 3 eq Zn sequentially to a centrifuge tube. 2+ After the solution has reacted fully for 240 min, 3.60 mL of methanol solution was added to bring the volume to 4 mL, and the fluorescence was measured.

[0084] The excitation wavelength and emission wavelength were set to 360 nm and 380–700 nm, respectively, so the final concentration of (R)-1 in the solution was 10 μM.

[0085] As shown in Figures 12(a), (b), and (c), probe (R)-1 does not exhibit fluorescence in methanol, but when 3 eq. Zn is added to (R)-1... 2+ After reacting with 30 eq. L-Trp, a significant fluorescence enhancement was observed at λ = 560 nm.

[0086] Within the range of 0.8–24.0 mM, the fluorescence intensity of L-Trp gradually increased with increasing concentration. Within the range of 24.0–120.0 mM, the fluorescence intensity of L-Trp began to decrease with increasing concentration. Therefore, in subsequent related tests, the concentration of L-Trp was set at 24 mM (30 eq).

[0087] Furthermore, the recognition process of (R)-1 for L-Trp was investigated in depth using UV-Vis spectroscopy, and the results are shown in Figures 13(a) and (b). As the L-Trp concentration gradually increased, the absorbance showed an increasing trend over a wide wavelength range. When the L-Trp concentration was below 1 equivalent, its main UV-Vis peaks remained at essentially the same wavelength, with only slight changes in intensity. With further increases in concentration, a significant redshift was observed from 225 nm to 300 nm, and the peaks at 250 nm and 280 nm also increased accordingly. These changes indicate a significant alteration in the conjugated system.

[0088] like Figure 14 As shown, during the titration process in the kinetic study, it was observed that when L-Trp was added, the aldehyde proton peak originally located at 10.20 ppm did not disappear rapidly, and the imine proton signal peak did not form. Therefore, it can be inferred that the reaction between (R)-1 and Zn... 2+ After forming a complex with amino acids, this non-emission excited-state photon transfer process becomes unavailable, leading to enhanced fluorescence.

[0089] Based on the detection results of high-resolution mass spectrometry, as shown in Figures 16(a) and (b), and the characterization results of UV-Vis and NMR, it can be inferred that L-Trp and the fluorescent probe (R)-1 did not form an imine, but rather exhibited interaction with each other through intermolecular forces and Zn. 2+ A stable compound is formed under coordination, thereby achieving fluorescent recognition, and in Figure 15 The structure of the product that may be generated after (R)-1 interacts with L-Trp was deduced.

[0090] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. A method for preparing a fluorescent probe that specifically and selectively recognizes L-tryptophan, characterized in that: Includes the following steps, S1, Weigh ( R )-6 was dissolved in ultra-dry DCM, the solution was cooled to 0°C, and DIPEA was added dropwise, followed by a reaction of 3 h; bromomethyl methyl ether was added dropwise, and the reaction was continued for 1 h. R )-6 is ( R )-BINOL; S2. Then, HCl was added at 0°C to quench the reaction; then, ethyl acetate was used for multiple extractions, and the resulting organic phases were combined; the mixture was dried with anhydrous Na2SO4 and filtered to remove impurities. S3. The solvent was removed by vacuum to obtain the crude product; further purification was performed by column chromatography to obtain a white solid product. R )-5, the ( R )-5 is 2'-(methoxymethoxy)-[1,1'-binaphthyl]-2-ol; S4, will ( R )-5 was fully dissolved in ultra-dry THF, and the solution was cooled to -78°C; then n-butyllithium was added dropwise, the temperature was restored to room temperature, and the reaction continued for 2 hours; the temperature was then cooled to 0°C again, DMF was added dropwise, and the temperature was restored to room temperature for 1 hour. S5. Cool to 0℃ again, add saturated NH4Cl to quench the reaction, and extract with ethyl acetate multiple times. Then combine the organic phases, dry with anhydrous Na2SO4, and filter to remove impurities. S6. The solvent was removed by vacuum to obtain the crude product; the crude product was further purified by column chromatography to obtain a yellow oily product. R )-4, the ( R )-4 is 2'-hydroxy-2-(methoxymethoxy)-[1,1'-binaphthyl]-3-carboxaldehyde; S7, will ( R Add 4 to a 250ml eggplant-shaped flask, evacuate and purge with nitrogen three times, add ultra-dry acetonitrile to dissolve completely; cool to 0℃ and add DIPEA dropwise, then restore to room temperature and react for 2 hours, cool to 0℃ again and add acryloyl chloride dropwise, and after the addition is complete, restore to room temperature and react for 3 hours; S8. Add Ce(SO4)2·4H2O and reflux for 8 hours, then extract multiple times with ethyl acetate and combine the resulting organic phases. S9. The product was dried over anhydrous Na2SO4 and filtered to remove impurities; the solvent was removed under reduced pressure to obtain the crude product; the crude product was further purified by column chromatography to obtain a yellow solid product. R )-1, the ( R )-1 is 3'-formyl-2'-hydroxy-[1,1'-binaphthyl]-2-yl ester of acrylate.

2. The method for preparing a fluorescent probe that specifically and selectively recognizes L-tryptophan according to claim 1, characterized in that: In step S3, the eluent used in column chromatography is a mixture of petroleum ether and ethyl acetate in a ratio of 40:1; in step S6, the eluent used in column chromatography is a mixture of petroleum ether and ethyl acetate in a ratio of 10:1; and in step S9, the eluent used in column chromatography is a mixture of petroleum ether and ethyl acetate in a ratio of 20:1.

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