A fluorescent probe for specifically recognizing lysine and a preparation method thereof

Abstract

The application discloses a kind of fluorescence probe and preparation method of specifically identifying lysine, and the chemical name of fluorescence probe is 3,3'-((((3-formyl-3'-(hydroxymethyl)-[1,1'-binaphthalene]-2,2'-diyl)bis(oxy))bis(methylene))dibenzaldehyde, and chemical structural formula is as follows: the fluorescence probe of the application can specifically identify D-Lys in 20 kinds of amino acids, and its enantiomer can specifically identify L-Lys, prove the chiral recognition effect of probe, and the probe has the advantages such as high sensitivity, high selectivity, fast response.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of chemical analysis detection, in particular to a fluorescent probe for specifically recognizing lysine and a preparation method thereof. BACKGROUND

[0002] Chirality refers to the property that an object cannot be superimposed on its mirror image, like the left and right hands of a person, which are symmetrical to each other but cannot be superimposed. Chirality is a basic attribute of nature, from small molecules of amino acids, to large molecules of DNA and proteins, to macroscopic world of morning glory vines, snail shells, and cosmic nebulae, chiral phenomena are ubiquitous. Among them, amino acids are not only the basic building blocks of important biological macromolecules of proteins, but also important precursors and important chiral sources of many chiral materials, chiral drugs and chiral catalysts. Amino acids play an important physiological role in the human body, for example, lysine, as one of the essential amino acids for the human body, has a positive nutritional significance in promoting human growth and metabolism, enhancing immunity, promoting nutrient absorption, anti-virus, and treating depression. Once people thought that only L-configuration amino acids existed in the human body, but with the continuous deepening of scientific research, it was found that D-configuration amino acids also exist in the human body and play an extremely important physiological role. For example, D-serine plays an important role in improving learning and memory function and delaying aging; D-alanine plays a very important role in hormone regulation. Abnormal content of amino acid enantiomers is closely related to some major diseases (such as Alzheimer's disease, cancer, diabetes, viral infection, brain disease, kidney disease, etc.), and is of great concern in clinical diagnosis and treatment research. In this case, it is of great significance to establish a specific detection method for amino acid enantiomers for the diagnosis and treatment of diseases.

[0003] Green and efficient enantiomer identification methods still face many challenges. First, traditional amino acid enantiomer recognition methods (such as HPLC, GC, CE, IR, etc.) generally have the disadvantages of complicated procedures, high cost, time-consuming, poor stability, etc. Compared with them, fluorescence detection method has the advantages of simple operation, good selectivity, low cost, simple operation, low cost, etc. This new detection method has played an important role in the analysis fields of chemistry, biology, medicine, food, etc. Therefore, it has very important research significance to use fluorescence detection method for the determination of amino acid enantiomers. SUMMARY

[0004] The present application aims to solve the defects of the prior art, and provides a fluorescent probe for specifically recognizing lysine and a preparation method thereof, which improves the accurate detection and analysis ability of lysine, provides technical means for clinical diagnosis and disease treatment, and provides data support for research in the fields of biomedicine, biochemistry, etc.

[0005] The technical problem of the present application is solved by the following technical scheme: a fluorescence probe for specifically identifying lysine, characterized in that the chemical name of the fluorescence probe is 3,3'-((((3-formyl-3'-(hydroxymethyl)-[1,1'-binaphthalen]-2,2'-diyl)bis(oxy))bis(methylene))dibenzaldehyde, and the chemical structural formula is as follows:

[0006]

[0007] The present application further discloses a preparation method of the fluorescence probe for specifically identifying lysine, comprising the following steps:

[0008] Step 1, synthesis of 2,2'-bis(methoxymethoxy)-1,1'-binaphthalene

[0009] Naphthalene and sodium hydride are respectively dissolved in a proper amount of ultradry tetrahydrofuran, the two solutions are uniformly mixed at a suitable temperature, and then the reaction is restored to room temperature; after a certain period of time, the reaction system is cooled to a suitable temperature, and then bromomethyl methyl ether is added dropwise; then the reaction is restored to room temperature overnight; under the condition of an ice water bath, ultrapure water is added to quench the reaction, and then ethyl acetate is used for extraction; the organic phases are combined, dried with anhydrous sodium sulfate, filtered, and then rotary evaporated; the product is purified by recrystallization with n-hexane to obtain white solid product 2,2'-bis(methoxymethoxy)-1,1'-binaphthalene;

[0010] Step 2, synthesis of 2,2'-bis(methoxymethoxy)-[1,1'-binaphthalen]-3,3'-dialdehyde

[0011] 2,2'-bis(methoxymethoxy)-1,1'-binaphthalene is dissolved in a proper amount of ultradry tetrahydrofuran, and then n-butyllithium is added dropwise at a suitable temperature; the reaction system changes from colorless to brown; the reaction is restored to room temperature; after a certain period of time, the reaction system is cooled to a suitable temperature, and then ultradry N,N-dimethylformamide is gradually added; the reaction system becomes a grayish white turbid liquid; the reaction is restored to room temperature; after a certain period of time, saturated ammonium chloride solution is added to quench the reaction under the condition of an ice water bath; then ethyl acetate is used for extraction; the organic phases are combined, dried with anhydrous sodium sulfate, filtered, and then rotary evaporated to obtain yellow solid product; the product is purified by column chromatography and vacuum dried to obtain 2,2'-bis(methoxymethoxy)-[1,1'-binaphthalen]-3,3'-dialdehyde;

[0012] Step 3, synthesis of 3'-(hydroxymethyl)-2,2'-bis(methoxymethoxy)-[1,1'-binaphthalen]-3-formaldehyde

[0013] 2,2'-bis(methoxymethoxy)-[1,1'-binaphthalen]-3,3'-diformaldehyde was dissolved in a mixed solvent of methanol and tetrahydrofuran, and sodium triacetoxyborohydride was added in batches. After being warmed to a suitable temperature for a certain period of time, the reaction was quenched by adding ultrapure water under an ice water bath, and then extracted with ethyl acetate. The combined organic phase was dried over anhydrous sodium sulfate, and then rotary evaporation was performed to obtain a yellow solid. Column chromatography was performed for purification, and then vacuum drying was performed to obtain the product 3'-(hydroxymethyl)-2,2'-bis(methoxymethoxy)-[1,1'-binaphthalen]-3-formaldehyde.

[0014] Step 4, synthesis of 2,2'-dihydroxy-3'-(hydroxymethyl)-[1,1'-binaphthalen]-3-formaldehyde

[0015] 3'-(hydroxymethyl)-2,2'-bis(methoxymethoxy)-[1,1'-binaphthalen]-3-formaldehyde was dissolved in 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 dichloromethane was used for extraction. The combined organic phase was dried over anhydrous sodium sulfate, filtered, and then rotary evaporation was performed to obtain the yellow solid target product 2,2'-dihydroxy-3'-(hydroxymethyl)-[1,1'-binaphthalen]-3-formaldehyde.

[0016] Step 5, synthesis of 3,3'-((((3-formyl-3'-(hydroxymethyl)-[1,1'-binaphthalen]-2,2'-diyl)bis(oxy))bis(methylene))dibenzaldehyde

[0017] 2,2'-dihydroxy-3'-(hydroxymethyl)-[1,1'-binaphthalen]-3-formaldehyde was dissolved in a suitable amount of anhydrous acetonitrile, and then anhydrous potassium carbonate and 3-bromomethylbenzaldehyde were added. After being warmed to a suitable temperature for a certain period of time, the solvent was rotary evaporated, and then extracted with ethyl acetate. The combined organic phase was dried over anhydrous sodium sulfate, filtered, and then rotary evaporation was performed to obtain a yellow solid. Column chromatography was performed for purification, and then vacuum drying was performed to obtain the light yellow target product 3,3'-((((3-formyl-3'-(hydroxymethyl)-[1,1'-binaphthalen]-2,2'-diyl)bis(oxy))bis(methylene))dibenzaldehyde.

[0018] Further preferably, in step 1, the suitable temperature is -5-5°C, the certain period of time is 1.5-2.5h, the recrystallization temperature is set to -20-0°C, the binaphthol is (R)-BINOL or (S)-BINOL, and the molar ratio of the binaphthol, sodium hydride and bromomethyl methyl ether is 1:6:3.

[0019] Further preferably, in step 2, the suitable temperature is -5-5℃, the reaction time is 0.5h-1.5h, the reaction system is changed into grey-white turbid liquid and returns to room temperature, the reaction time is 2.5-3.5h, the molar ratio of 2,2'-bis(methoxymethoxy)-1,1'-binaphthalene: n-butyllithium: N-N dimethylformamide is 1:2.5:2.4, and the elution phase volume ratio of petroleum ether: ethyl acetate for column chromatography purification is 10:1.

[0020] Further preferably, in step 3, the suitable temperature is 60℃, the certain time is overnight, the volume ratio of tetrahydrofuran: methanol is 2:1, and the elution phase volume ratio of petroleum ether: ethyl acetate for column chromatography purification is 2:1.

[0021] Further preferably, in step 4, the certain time is overnight, and the volume ratio of dichloromethane: anhydrous ethanol is 1:1.

[0022] Further preferably, in step 5, the suitable temperature is 80℃, the certain time is overnight, and the elution phase volume ratio of petroleum ether: ethyl acetate for column chromatography purification is 3:1.

[0023] In summary, the present application has the following beneficial effects: the present application provides a fluorescent probe for specifically recognizing lysine and a preparation method thereof, which has the advantages of simple synthesis method, suitability for high-throughput analysis, low detection cost, good reaction condition, convenient post-treatment and the like. The aldehyde hydroxyl bifunctional fluorescent probe (S)-5 prepared by the present application and taking binaphthol (BONOL) as a skeleton can specifically recognize D-Lys among 20 kinds of chiral amino acids, while its enantiomer (R)-5 probe can specifically recognize L-Lys, indicating that the probe has chiral recognition effect. More importantly, the probe can simultaneously determine the concentration and enantiomer composition of lysine. The chiral lysine is chemically and enantioselectively recognized in a methanol solution, a new method for simultaneously determining the concentration and enantiomer composition of lysine is provided, and the method has high-throughput analysis potential. The detection method has short response time and high sensitivity, meets the requirement of rapid detection of chiral amino acids, and has important application value in many fields. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Chemical structural formulas of the two kinds of chiral lysine;

[0025] Figure 2 Synthesis route diagram of the fluorescent probe (S)-5 for specifically recognizing lysine;

[0026] Figure 3 Nuclear magnetic hydrogen spectrum diagram of the compound (S)-5;

[0027] Figure 4 NMR spectrum of compound (S)-5;

[0028] Figure 5 Column chart of fluorescence intensity at 450 nm after reaction of fluorescent probe (S)-5 with 20 kinds of chiral amino acids;

[0029] Figure 6 Correspondence diagram of fluorescence intensity at 450 nm of fluorescent probe (S)-5 and (R)-5 with the composition (ee value) of lysine enantiomers;

[0030] Figure 7 Test research result diagram of the influence of reaction time on the specific recognition of lysine. Figure 7 (a) is the fluorescence spectrum diagram of fluorescent probe (S)-5 and D-Lys under different reaction times; Figure 7 (b) is the fluorescence spectrum diagram of fluorescent probe (S)-5 and L-Lys under different reaction times; Figure 7 (c) is the change trend diagram of fluorescence intensity at 450 nm after reaction of fluorescent probe (S)-5 and D / L-Lys under different reaction times;

[0031] Figure 8 Test research result diagram of the influence of lysine concentration on the specific recognition of lysine. Figure 8 (a) is the fluorescence spectrum diagram of fluorescent probe (S)-5 after reaction with different equivalent D-Lys; Figure 8 (b) is the fluorescence spectrum diagram of fluorescent probe (S)-5 after reaction with different equivalent L-Lys; and Figure 8 (c) is the change trend diagram of fluorescence intensity at 450 nm after reaction of fluorescent probe (S)-5 and different equivalent D / L-Lys;

[0032] Figure 9 Test research result diagram of the influence of Zn 2+ concentration on the specific recognition of lysine; Figure 9 (a) is the fluorescence spectrum diagram of fluorescent probe (S)-5 and D-Lys after reaction under different Zn 2+ concentrations; in Figure 9 (b) is the fluorescence spectrum diagram of fluorescent probe (S)-5 and L-Lys after reaction under different Zn 2+ concentrations; Figure 9 (c) is the change trend diagram of fluorescence intensity at 450 nm after reaction of fluorescent probe (S)-5 and D / L-Lys under different Zn 2+ concentrations. DETAILED DESCRIPTION

[0033] The application will be further described in detail below in combination with the drawings.

[0034] 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 conjunction with examples. Those skilled in the art will understand that the following examples are only for illustrating the present application and should not be regarded as limiting the scope of the present application.

[0035] The chiral fluorescent probe is divided into S and R two configurations, and the lysine to be specifically recognized by the present application is divided into D and L two configurations, Figure 1 The chemical structural formulas of the two chiral lysines are as shown in the following formula (I) and formula (II). Figure 1 As shown in the formula (I) and formula (II), the chiral lysine includes D-lysine (D-Lys) and L-lysine (L-Lys).

[0036] Example 1

[0037] Figure 2 The synthesis route of the fluorescent probe for specifically recognizing the chiral lysine prepared in the present application is as shown in the following formula (III), which includes the following steps: Figure 2

[0038] Step 1. Synthesis of 2,2'-bis(methoxymethoxy)-1,1'-binaphthyl (S)-1

[0039] (S)-BINOL (7.5 g, 26.25 mmol) and sodium hydride (6.3 g, 157 mmol) were weighed and dissolved in 50 mL of ultra-dry tetrahydrofuran, respectively, and the two solutions were mixed uniformly at 0°C, and then the reaction was restored to room temperature; after 2 h of reaction, the reaction system was cooled to 0°C, and bromomethyl methyl ether (6.5 mL, 78.3 mmol) was added dropwise, and then the reaction was restored to room temperature overnight; under the condition of ice water bath, the reaction was quenched by adding ultra-pure water, and then extracted with ethyl acetate, and the organic phase was combined and dried with anhydrous sodium sulfate, and then filtered, and then rotary evaporated, and then the product was purified by recrystallization with n-hexane to obtain 9.83 g of white solid product (S)-1, with a yield of 91.9%.

[0040] ​Step 2. Synthesis of 2,2'-bis(methoxymethoxy)-[1,1'-binaphthalen]-3,3'-diformaldehyde (S)-2 The white solid product (S)-1 (4 g, 10.68 mmol) was dissolved in 45 mL of super dry tetrahydrofuran, and n-butyllithium (16.7 mL, 26.7 mmol) was added dropwise at 0 °C. The reaction system changed from colorless to brown, and was restored to room temperature for 2 h. The reaction system was cooled to 0 °C again, and super dry N,N-dimethylformamide (2 mL, 25.63 mmol) was added dropwise. The reaction system changed to a grayish white turbid liquid, and was restored to room temperature for 3 h. After the reaction was completed, 100 mL of saturated ammonium chloride solution was added to quench the reaction under ice water bath conditions. Then extraction was performed with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain a yellow solid product. Column chromatography was used for purification, and the eluent was petroleum ether: ethyl acetate = 10:1 by volume. After vacuum drying, 4.3 g of product (S)-2 was obtained with a yield of 67.0%.

[0041] Step 3. Synthesis of 3'-(hydroxymethyl)-2,2'-bis(methoxymethoxy)-[1,1'-binaphthalen]-3- formaldehyde (S)-3

[0042] The product (S)-2 (430.5 mg, 1 mmol) was dissolved in 10 mL of methanol and 20 mL of super dry tetrahydrofuran mixed solvent, and sodium triacetoxyborohydride (211.9 mg, 1 mmol) was added in batches. After being warmed to 60 °C, the reaction was performed overnight. Then 10 mL of super pure water was added to quench the reaction under ice water bath conditions, and extraction was performed with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and rotary evaporated to obtain a yellow solid. Column chromatography was used for purification, and the eluent was petroleum ether: ethyl acetate = 2:1 by volume. After vacuum drying, 215 mg of yellow solid product (S)-3 was obtained with a yield of 49.7%.

[0043] Step 4. Synthesis of 2,2'-dihydroxy-3'-(hydroxymethyl)-[1,1'-binaphthalen]-3-formaldehyde (S)-4

[0044] The product (S)-3 (200 mg, 0.46 mmol) was dissolved in 20 mL of dichloromethane and 20 mL of anhydrous ethanol mixed solvent, and 1 mL of concentrated hydrochloric acid was added dropwise. After a certain period of time, sodium bicarbonate was added to quench the reaction, and extraction was performed with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain 157.4 mg of yellow solid target product (S)-4 with a yield of 99%.

[0045] Step 5. Synthesis of 3,3'-((((3-formyl-3'-(hydroxymethyl)-[1,1'-binaphthalen]-2,2'- diyl)bis(oxy))bis(methylene))dibenzaldehyde (S)-5

[0046] The product (S)-4 (155 mg, 0.45 mmol) was dissolved in 20 mL of anhydrous acetonitrile, and then anhydrous potassium carbonate (497 mg, 3.6 mmol) and 3-bromomethylbenzaldehyde (188 mg, 0.95 mmol) were added, and the temperature was raised to 80°C. After the reaction was carried out overnight, the solvent was evaporated, and then extracted with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain a yellow solid, which was purified by column chromatography, and then dried in vacuum to obtain 210 mg of the target product (S)-5 in a light yellow color, with a yield of 80.6%.

[0047] The nuclear magnetic hydrogen spectrum and the nuclear magnetic carbon spectrum of the compound (S)-5 prepared in this example were as follows: Figure 3 、 Figure 4 .

[0048] The preparation method provided by the example of the present application has simple reaction devices, mild reaction conditions, and low cost.

[0049] Example 2

[0050] The difference between this example and Example 1 is that (S)-BINOL is replaced by (R)-BINOL, and finally the product (R)-5 is prepared.

[0051] Example 3

[0052] The method for detecting 20 kinds of amino acids in methanol solution by using the fluorescent probe prepared in Example 1 comprises the following steps:

[0053] Preparation of probe solution: (S)-5 is dissolved in an appropriate amount of chromatographic grade methanol to obtain a 1.6 mM probe solution;

[0054] Preparation of amino acid solution: 20 kinds of amino acids are respectively dissolved in ultrapure water to prepare 1-10 eq of an amino acid solution;

[0055] Preparation of zinc acetate solution: zinc acetate is dissolved in ultrapure water to prepare a 0.5-8 eq zinc acetate solution;

[0056] Subsequently, 50 uL of the probe solution, 250 uL of methanol, 50 uL of the amino acid solution, and 50 uL of the zinc acetate solution are sequentially added to a centrifuge tube, and after 1 h of reaction, 3.6 mL of methanol is added to make the volume 4 mL, and then the mixture is shaken and subjected to fluorescence testing, wherein the fluorescence testing parameters are: the excitation light slit is 5 nm, the emission light slit is 5 nm, and the excitation light wavelength is 320 nm.

[0057] Example 4

[0058] The difference between this example and Example 3 is that the compound (R)-5 prepared in Example 1 is used to detect 20 kinds of amino acids in methanol.

[0059] AsFigure 5 As shown, under the excitation wavelength of 320 nm, the fluorescence probe (S)-5 + 1 eq Zn 2+ When interacting with 20 kinds of amino acids in methanol solution, D-Lys can make the fluorescence probe produce strong fluorescence emission at λ = 450 nm, while L-Lys and other 19 kinds of amino acid enantiomers have almost no fluorescence enhancement, which shows that the fluorescence probe can specifically recognize D-Lys among 20 kinds of amino acids. However, in the presence of Zn 2+ , the fluorescence intensity of (S)-5 probe and its enantiomer (R)-5 probe after reacting with lysine at λ = 450 nm has a mirror relationship, and at the same time, the fluorescence intensity of (R)-5 + Zn 2+ after reacting with 20 kinds of amino acids is measured, which confirms that (R)-5 can specifically recognize L-Lys among 20 kinds of amino acids.

[0060] Two probe enantiomers (S)-5 and (R)-5 + Zn 2+ The fluorescence intensity at 450 nm has a mirror relationship with the ee value of D-Lys [ee = ([D] - [L]) / ([D] + [L])], as shown in FIG. 6, with the increase of the component ratio of D-Lys, the fluorescence intensity of (S)-5 probe at 450 nm shows a linear growth trend, while the fluorescence intensity of (R)-5 at 450 nm decreases regularly, which shows that the probe can be used to measure the enantiomeric composition of lysine.

[0061] Therefore, the present application realizes the specific fluorescence recognition of lysine among 20 kinds of amino acids, provides a new method for rapidly detecting the enantiomeric concentration and composition of lysine, and has the potential of high-throughput analysis.

[0062] Example 5 The influence factors and principles of the fluorescence probe recognizing lysine in the present application are explored.

[0063] Test and research on the influence of reaction time on specific recognition of lysine

[0064] 50 μL of mother liquor was taken from the standard sample of (S)-5 and transferred to a clean 5 mL centrifuge tube, and then 250 μL of methanol solution was taken. Subsequently, 50 μL of 9.6 mM (6 eq) D-Lys or L-Lys sample to be tested was taken into the centrifuge tube. At the same time, 50 μL of 1.6 mM (1 eq) zinc acetate solution was quickly taken into the centrifuge tube. After slight shaking and mixing, the reaction was carried out at room temperature for 1 min, 10 min, 20 min, 30 min, 40 min, 50 min, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, and 7 h, respectively. After the reaction was completed, 3.6 mL of chromatographic grade methanol was added to dilute the reaction solution to 4 mL. Immediately after dilution, the solution was transferred to a cuvette for fluorescence spectrum test. The results are referred to Figure 7 ,Figure 7 (a) Probe (S)-5 + Zn 2+ Fluorescence spectra of probe (S)-5 + Zn with D-Lys at different reaction time. In Figure 7 (b) Probe (S)-5 + Zn 2+ Fluorescence spectra of probe (S)-5 + Zn with L-Lys at different reaction time. In Figure 7 (c) Probe (S)-5 + Zn 2+ The trend of fluorescence intensity at 450 nm after the reaction of probe (S)-5 + Zn with D / L-Lys at different Lys concentration. As can be seen from the figure, the fluorescence intensity increases significantly before 1 h, and then increases slowly. In order to meet the demand of rapid detection and ensure practicability, we set the reaction time to 1 h.

[0065] Test research on the effect of lysine concentration on the specific recognition of lysine

[0066] Take 50 μL mother liquor from the standard sample of (S)-5, and transfer it to a clean 5 mL centrifuge tube. Then take 250 μL of chromatographic grade methanol solution. Subsequently, take 50 μL of 1.6 mM, 3.2 mM, 4.8 mM, 6.4 mM, 8.0 mM, 9.6 mM, 11.2 mM, 12.8 mM, 14.4 mM and 16.0 mM L-Lys or D-Lys into the centrifuge tube, respectively. At the same time, quickly take 50 μL of zinc acetate solution into the centrifuge tube, mix gently, and then stand for 1 h after the reaction. Add 3.6 mL of chromatographic grade methanol to make the reaction solution to 4 mL. Immediately after dilution, transfer the solution to a cuvette for fluorescence spectrum test. The results are referred to Figure 8 Figure 8 (a) Probe (S)-5 + Zn 2+ Fluorescence spectra of probe (S)-5 + Zn after 1 h reaction with different equivalent of D-Lys. In Figure 8 (b) Probe (S)-5 + Zn 2+ Fluorescence spectra of probe (S)-5 + Zn after 1 h reaction with different equivalent of L-Lys. In Figure 8 (c) Probe (S)-5 + Zn 2+ The trend of fluorescence intensity at 450 nm after the reaction of probe (S)-5 + Zn with D / L-Lys at different Lys concentration. As can be seen from the figure, the fluorescence intensity increases first and then decreases with the increase of D-Lys equivalent, and reaches the maximum value at 6 eq. With the increase of L-Lys equivalent, the fluorescence intensity remains at a low level and does not change significantly. Therefore, the optimal equivalent of lysine is determined to be 6 eq.

[0067] Zn 2+ Test research on the effect of zinc concentration on the specific recognition of lysine

[0068] ​Take 50 μL mother liquor from the standard sample of (S)-5 and transfer to a clean 5 mL centrifuge tube, then take 250 μL chromatographic grade methanol solution. Then, take 50 μL 9.6 mM (6 equivalents) D-Lys or L-Lys to the centrifuge tube. At the same time, quickly take 50 μL 0.8 mM, 1.6 mM, 3.2 mM, 4.8 mM, 6.4 mM, 8.0 mM, 9.6 mM, 11.2 mM and 12.8 mM zinc acetate solution to the centrifuge tube. After mixing by slight shaking, after 1 h of standing at room temperature, add 3.6 mL chromatographic grade methanol to dilute the reaction solution to 4 mL. Immediately after dilution, transfer the solution to a cuvette for fluorescence spectrum test. The results are shown in Figure 9 , Figure 9 (a) is the fluorescence spectrum of probe (S)-5 after 1 h of reaction with D-Lys at different equivalents of Zn 2+ Figure 9 (b) is the fluorescence spectrum of probe (S)-5 after 1 h of reaction with L-Lys at different equivalents of Zn 2+ Figure 9 (c) shows the change trend of the fluorescence intensity at 450 nm of probe (S)-5 after 1 h of reaction with D / L-Lys at different equivalents of Zn 2+ As can be seen from the figure, the fluorescence intensity first increases and then decreases with the increase of the equivalent of D-Lys, and reaches the maximum value at 1 eq. With the increase of the equivalent of L-Lys, the fluorescence intensity remains at a low level and does not change significantly. Therefore, the optimal equivalent of Zn 2+ is determined to be 1 eq.

[0069] The specific embodiment is only an explanation of the present application, which is not a limitation of the present application. Those skilled in the art can make modifications to the embodiment without creative contribution after reading the specification, but as long as it is within the scope of the claims of the present application, it is protected by the patent law.​​

Claims

1. A fluorescent probe that specifically recognizes lysine, characterized in that, The fluorescent probe is chemically named 3,3'-((((3-formyl-3'-(hydroxymethyl)-[1,1'-binaphthyl]-2,2'-diyl)bis(oxy))bis(methylene))dibenzaldehyde, and its chemical structure is as follows: .

2. A method for preparing a fluorescent probe that specifically recognizes lysine as described in claim 1, characterized in that, Includes the following steps: Step 1: Synthesis of 2,2'-bis(methoxymethoxy)-1,1'-binaphthyl S-binaphthol and sodium hydride were dissolved separately in an appropriate amount of ultra-dry tetrahydrofuran. The two solutions were mixed evenly under suitable temperature conditions, and then the reaction was allowed to proceed at room temperature. After a certain period of time, the reaction system was cooled to a suitable temperature, and bromomethyl methyl ether was added dropwise. The reaction was then allowed to proceed at room temperature overnight. The reaction was quenched with ultrapure water under ice-water bath conditions, and then extracted with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and evaporated by rotary evaporation. The product was then purified by recrystallization with n-hexane to obtain a white solid product 2,2'-bis(methoxymethoxy)-1,1'-binaphthol. Step 2: Synthesis of 2,2'-bis(methoxymethoxy)-[1,1'-binaphthyl]-3,3'-dicarboxaldehyde 2,2'-bis(methoxymethoxy)-1,1'-binaphthylene was dissolved in an appropriate amount of ultra-dry tetrahydrofuran. Under suitable temperature conditions, n-butyllithium was added dropwise, and the reaction system changed from colorless to brown. The reaction was then restored to room temperature. After a certain period of time, the reaction system was cooled to a suitable temperature, and ultra-dry N,N-dimethylformamide was gradually added. The reaction system turned into a grayish-white turbid liquid. The reaction was then restored to room temperature. After a certain period of time, the reaction was quenched by adding saturated ammonium chloride solution under ice-water bath conditions. The mixture was then extracted with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain a yellow solid product. The product was purified by column chromatography and dried under vacuum to obtain the product 2,2'-bis(methoxymethoxy)-[1,1'-binaphthylene]-3,3'-dicarboxaldehyde. Step 3: Synthesis of 3'-(hydroxymethyl)-2,2'-bis(methoxymethoxy)-[1,1'-binaphthyl]-3-carboxaldehyde 2,2'-bis(methoxymethoxy)-[1,1'-binaphthyl]-3,3'-dicarboxaldehyde was dissolved in a mixed solvent of methanol and tetrahydrofuran. Triacetoxysodium borohydride was added in portions. After heating to a suitable temperature, the reaction was carried out for a certain period of time. The reaction was quenched with ultrapure water in an ice-water bath. The mixture was then extracted with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and rotary evaporated to obtain a yellow solid. The solid was purified by column chromatography and dried under vacuum to obtain the product 3'-(hydroxymethyl)-2,2'-bis(methoxymethoxy)-[1,1'-binaphthyl]-3-carboxaldehyde. Step 4: Synthesis of 2,2'-dihydroxy-3'-(hydroxymethyl)-[1,1'-binaphthyl]-3-carboxaldehyde 3'-(hydroxymethyl)-2,2'-bis(methoxymethoxy)-[1,1'-binaphthyl]-3-carboxaldehyde was dissolved in a suitable amount of dichloromethane and anhydrous ethanol. Concentrated hydrochloric acid was added dropwise. After a certain period of time, sodium bicarbonate was added to quench the reaction. The mixture was then extracted with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain the yellow solid target product 2,2'-dihydroxy-3'-(hydroxymethyl)-[1,1'-binaphthyl]-3-carboxaldehyde. Step 5: Synthesize 3,3'-((((3-formyl-3'-(hydroxymethyl)-[1,1'-binaphthyl]-2,2'-diyl)bis(oxy))bis(methylene))dibenzaldehyde 2,2'-Dihydroxy-3'-(hydroxymethyl)-[1,1'-binaphthyl]-3-carboxaldehyde was dissolved in an appropriate amount of anhydrous acetonitrile. Anhydrous potassium carbonate and 3-bromomethylbenzaldehyde were added, and the mixture was heated to a suitable temperature. After reacting for a certain period of time, the solvent was evaporated, and the mixture was extracted with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain a yellow solid. The solid was purified by column chromatography and dried under vacuum to obtain the pale yellow target product 3,3'-((((3-formyl-3'-(hydroxymethyl)-[1,1'-binaphthyl]-2,2'-diyl)bis(oxy))bis(methylene))dibenzaldehyde.

3. The method for preparing a fluorescent probe that specifically recognizes lysine according to claim 2, characterized in that, In step 1, the suitable temperature is -5 to 5°C, the certain time is 1.5 to 2.5 h, the recrystallization temperature is set to -20 to 0°C, and the molar ratio of S-binaphthol: sodium hydride: bromomethyl methyl ether is 1:6:

3.

4. The method for preparing a fluorescent probe that specifically recognizes lysine according to claim 2, characterized in that, In step 2, the suitable temperature is -5 to 5°C. The reaction time is 0.5 to 1.5 hours after the reaction system changes from colorless to brown and returns to room temperature. The reaction time is 2.5 to 3.5 hours after the reaction system becomes a grayish-white turbid liquid and returns to room temperature. The molar ratio of 2,2'-bis(methoxymethoxy)-1,1'-binaphthyl: n-butyllithium: N,N-dimethylformamide is 1:2.5:2.

4. The volume ratio of the eluent phase for column chromatography purification is petroleum ether: ethyl acetate = 10:

1.

5. The method for preparing a fluorescent probe that specifically recognizes lysine according to claim 2, characterized in that, In step 3, the suitable temperature is 60°C, the certain time is overnight, the volume ratio of tetrahydrofuran to methanol is 2:1, and the volume ratio of the eluent phase for column chromatography purification is petroleum ether to ethyl acetate = 2:

1.

6. The method for preparing a fluorescent probe that specifically recognizes lysine according to claim 2, characterized in that, In step 4, the specified time is overnight, and the volume ratio of dichloromethane to anhydrous ethanol is 1:

1.

7. The method for preparing a fluorescent probe that specifically recognizes lysine according to claim 2, characterized in that, In step 5, the suitable temperature is 80°C, the certain time is overnight, and the volume ratio of the eluent phase for column chromatography purification is petroleum ether: ethyl acetate = 3:1.

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