Preparation of a fluorescent probe for recognizing chiral glucose and detection method thereof

Abstract

The application discloses a preparation and detection method of a fluorescent probe for recognizing chiral glucose and belongs to the technical field of fluorescence detection. (R)-6 is weighed and dissolved in DCM, DIPEA and bromomethyl methyl ether are added dropwise, HCl is added at 0 DEG C for quenching, and organic phases are extracted and combined; a crude product is obtained by removing the solvent under reduced pressure, and (R)-5 is obtained by purification; (R)-5 is dissolved in THF, and n-butyllithium and DMF are added dropwise after cooling; NH4Cl is added for quenching, organic phases are extracted and combined after extraction, and drying treatment is performed; a crude product is obtained by removing the solvent under reduced pressure, and (R)-4 is obtained by purification; (R)-4 and potassium carbonate are dissolved in acetonitrile, and 4-(bromomethyl)phenylboronic acid is added; after extraction and combination of organic phases, drying treatment is performed, a crude product is obtained by removing the solvent under reduced pressure, and (R)-2 is obtained by purification; (R)-2 is dissolved in dichloromethane and stirred, HCl is added to a flask, pure water and NaHCO3 are added; organic phases are extracted and combined and drying treatment is performed, the solvent is removed through reduced pressure operation, and (R)-3 is obtained. The application realizes specific and selective recognition of D-glucose, and provides a brand-new idea for developing a probe for glucose detection in the field of biological correlation.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of fluorescence detection, and particularly relates to a preparation and detection method of a fluorescence probe for recognizing chiral glucose. BACKGROUND

[0002] Glucose has important significance in the fields of biology, medicine and food industry, and is one of the indispensable substances for maintaining life activities. Glucose is the most widely distributed and most important monosaccharide in nature, which is the energy source of living cells and the intermediate product of metabolism, i.e. the main energy-providing substance of organisms. Plants can produce glucose through photosynthesis, while animals and humans obtain glucose by ingesting food to meet the needs of life activities. Glucose has a wide range of applications in medicine. First, it can supplement energy for patients, especially for patients with insufficient energy intake, coma, shock or dehydration. When patients have severe vomiting, diarrhea or blood loss, glucose can play a role in supplementing water and heat. In addition, glucose can also be used to treat hypoglycemia symptoms such as dizziness, blurred vision, etc. Oral or intravenous injection of glucose can quickly correct the state of hypoglycemia. At the same time, glucose is also used as a solvent and diluent for injection and oral drugs, and a raw material for manufacturing medical insulin, playing an important role in the treatment of diabetes. Glucose also has a wide range of applications in the food industry. It can be used as a sweetener for seasoning and saccharification of food to improve the taste of food. At the same time, glucose can also be used as a food additive to improve the moisture retention and color stability of food. In addition, glucose is also an important fermentation substrate and is widely used in food fermentation processes, such as manufacturing yeast bread, yogurt and other foods. Therefore, it is particularly important to recognize glucose technology. Through specific and selective recognition, we can accurately distinguish this amino sugar, thereby ensuring its safety and effectiveness in different application fields. This is not only crucial for the development of the chemical industry, but also provides a new perspective and tool for research in the field of life sciences. Fluorescence detection technology is selected as the detection means because of its high sensitivity, high selectivity and high-throughput analysis. In this work, a new type of fluorescence probe (S)-5 with multiple reaction sites of aldehyde, hydroxyl and boronic acid groups based on BINOL skeleton was designed and synthesized, and specific recognition of D-glucose in methanol was successfully achieved.

[0003] Although there are examples of fluorescent probes that can selectively recognize glucose, the total amount of these probes is relatively small, and their selective recognition effect is not ideal. In order to overcome these limitations, we plan to use new strategies to improve the total amount and selective recognition ability of the probes. In previous research work, Javier et al. designed a new type of fluorescent monosaccharide receptor that works in aqueous media, which is characterized by a VIE signal unit connected to two phenylboronic acid motifs through an aminomethyl moiety, which can form a dynamic covalent bond with 1,2-diol. This system can distinguish very similar compounds such as monosaccharides D-glucose, D-galactose and D-fructose, causing a significant change in fluorescence that can be monitored with the naked eye. In the case of glucose, even in the presence of other monosaccharides, the recognition process causes the most dramatic change in color and fluorescence intensity. Considering the constraints imposed by the formation of a dynamic covalent bond between the two phenylboronic acid moieties and the monosaccharide, the vibration-induced planarization of the excited state of the fluorophore will be hindered, resulting in a low color shift observed in the emission. Therefore, we plan to take advantage of the excellent BINOL skeleton by adjusting the spatial size of the linking group to further increase the rigidity of the probe to achieve efficient and specific selective recognition of different monosaccharides, especially glucose. SUMMARY

[0004] Therefore, the present application aims to provide a preparation and detection method of a fluorescent probe for recognizing chiral glucose.

[0005] To achieve the above-mentioned purpose, the present application provides the following technical solutions.

[0006] The present application provides a preparation and detection method of a fluorescent probe for recognizing chiral glucose, comprising the following steps,

[0007] S1, weigh (R)-6 and dissolve it in super-dry DCM, then drop DIPEA into the solution after cooling to 0℃, and react for 3h; then slowly drop bromomethyl methyl ether into the solution, and react for 1h;

[0008] S2, add 2M HCl at 0℃ to quench the reaction, then extract with ethyl acetate, combine the organic phase, dry and filter to remove impurities, then remove the solvent under reduced pressure to obtain the crude product, and purify the crude product by column chromatography to obtain white solid product (R)-5;

[0009] S3, dissolve (R)-5 in super-dry THF, then slowly drop n-butyllithium into the solution after cooling to-78℃, and react for 2h at room temperature; then slowly drop DMF into the solution after cooling to 0℃ again, and react for 1h at room temperature;

[0010] S4, quenching the reaction by adding saturated NH4Cl at 0℃, and extracting with ethyl acetate, drying and treating the combined organic phase, and filtering to remove impurities; then removing the solvent by reduced pressure to obtain a crude product; purifying the crude product by column chromatography to obtain a yellow oil product (R)-4;

[0011] S5, dissolving (R)-4 and potassium carbonate in 30 mL of acetonitrile, stirring at 50℃ for 3h, and then adding 4-(bromomethyl)phenylboronic acid and reacting for 21h;

[0012] S6, after the reaction is completed, washing the residue with acetonitrile, extracting with ethyl acetate, drying and treating the combined organic phase, and filtering to remove impurities; then removing the solvent by reduced pressure to obtain a crude product; purifying the crude product by column chromatography to obtain a yellow product (R)-2;

[0013] S7, dissolving (R)-2 (0.95 mmol, 468mg) in dichloromethane and stirring uniformly, and then adding 1ml of HCl in a flask;

[0014] S8, continuously tracking the reaction until only one product spot is obtained, at which time adding ultrapure water to the flask, and then adding NaHCO3 solid multiple times until the bubbles disappear;

[0015] S9, extracting three times with ethyl acetate, drying and treating the combined organic phase, and filtering to remove impurities, and then removing the solvent by reduced pressure to obtain a yellow product (R)-3.

[0016] Further, in the steps S2, S4, S6 and S9, the drying treatment is performed by using anhydrous Na2SO 4。

[0017] Further, in the step S2, when purifying the crude product by column chromatography, the eluent is prepared by using petroleum ether and ethyl acetate in a ratio of 40:1; in the step S4, when purifying the crude product by column chromatography, the eluent is prepared by using petroleum ether and ethyl acetate in a ratio of 10:1; and in the step S6, when purifying the crude product by column chromatography, the eluent is prepared by using petroleum ether and ethyl acetate in a ratio of 30:1.

[0018] The present application has the following beneficial effects:

[0019] 1. The present application realizes specific and selective recognition of D-glucose based on the characteristics of fluorescent substances and the interaction between D-glucose and specific fluorescent markers or probes.

[0020] 2. Selective recognition of fluorescent markers or probes of the present invention: By designing and selecting fluorescent markers or probes that have specific selective recognition of D-glucose, these markers or probes can bind to D-glucose to form a stable complex and generate a detectable fluorescent signal.

[0021] 3. The principle of fluorescence resonance energy transfer (FRET) in this invention: Under certain conditions, the principle of fluorescence resonance energy transfer can be used to achieve specific and selective recognition of D-glucose. FRET is a distance-dependent nonradiative energy transfer phenomenon. When the emission spectrum of a fluorescent donor overlaps with the absorption spectrum of a fluorescent acceptor, and the distance between the two molecules is within 10 nm, a nonradiative energy transfer, i.e., the FRET phenomenon, occurs. By designing specific fluorescent donors and acceptors, the distance or conformation between the donor and acceptor will change when D-glucose is present, thereby affecting the occurrence of the FRET phenomenon and thus achieving specific and selective recognition of D-glucose.

[0022] 4. Fluorescence quenching or enhancement effect of the present invention: When D-glucose binds to a fluorescent label or probe, it may alter the fluorescence properties of the fluorescent label or probe, such as fluorescence intensity and fluorescence lifetime. By measuring these changes in fluorescence properties, specific and selective recognition of D-glucose can be achieved.

[0023] 5. This invention provides a novel approach for developing probes for glucose detection in biological fields, and opens up new avenues for the design and optimization of fluorescent probes.

[0024] 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

[0025] To make the purpose, technical solution, and beneficial effects of the invention clearer, the following figures are provided for illustration:

[0026] Figure 1 This is a synthetic route diagram of the present invention;

[0027] Figure 2 The probe of this invention ( R -5 1 H NMR spectrum;

[0028] Figure 3 The probe of this invention ( R -5 13 C NMR spectrum;

[0029] Figure 4 The probe of this invention ( R )-4 1 H NMR spectrum;

[0030] Figure 5 The probe of this invention ( R )-4 13 C NMR spectrum;

[0031] Figure 6 The probe of this invention ( R )-2 1 H NMR spectrum;

[0032] Figure 7 The probe of this invention ( R )-2 13 C NMR spectrum;

[0033] Figure 8 The probe of this invention ( R )-3 1 H NMR spectrum;

[0034] Figure 9 The probe of this invention ( R )-3 13 C NMR spectrum;

[0035] Figure 10 The probe (S)-3 of this invention 1 H NMR spectrum;

[0036] Figure 11 The image shows the detection results of the probe (R)-3 of this invention for L-glucose and D-glucose.

[0037] Figure 12 The image shows the detection results of the probe (S)-3 of this invention for L-glucose and D-glucose in methanol solution.

[0038] Figure 13 The image shows the detection results of the probe (S)-3 of this invention on 17 amino sugars in methanol solution. Detailed Implementation

[0039] A method for preparing and detecting a fluorescent probe for recognizing chiral glucose.

[0040] Figure 1 This is a synthetic route diagram of the fluorescent probe for recognizing chiral glucose (referred to as probe (R)-3 / (S)-3) in this invention.

[0041] The glucose used in the detection process of this invention is commercially available glucose.

[0042] Example 1

[0043] A method for preparing a fluorescent probe for recognizing chiral glucose, comprising the following steps:

[0044] S1, weigh (R)-6 (17.5 mmol, 5 g), that is, (R)-BINOL, and dissolve it in 100 mL of ultra-dry DCM; slowly drop DIPEA (38.5 mmol, 6.4 mL) into the solution cooled to 0°C, and react for 3 h; then slowly drop bromomethyl methyl ether (26.2 mmol, 2.1 mL) into the solution, and react for 1 h;

[0045] S2, add 2M HCl (50 mL) into the solution at 0°C to quench the reaction, then extract the solution with 20 mL of ethyl acetate for 3 times, combine the organic phases, dry the combined organic phases by using anhydrous Na2SO4, filter to remove impurities, remove the solvent by reducing pressure, and obtain a crude product; purify the crude product by column chromatography, wherein the eluent is prepared by using petroleum ether and ethyl acetate in a ratio of 40:1, and obtain 4.0 g of white solid product (R)-5, that is, 2'-(methoxymethoxy)-[1,1'-binaphthalen]-2-ol, with a yield of 80%, and its 1 H NMR is shown in Figure 2 , 13 C NMR is shown in Figure 3 ;

[0046] S3, dissolve (R)-5 (12.2 mmol, 4 g) in 100 mL of ultra-dry THF, cool the solution to -78°C, slowly drop n-butyllithium (42.8 mmol, 26.8 mL, 1.6M dissolved in n-hexane) into the solution, restore the solution to room temperature, and react for 2 h; cool the solution to 0°C again, slowly drop DMF (18.4 mmol, 1.44 mL) into the solution, restore the solution to room temperature, and react for 1 h;

[0047] S4, add 20 mL of saturated NH4Cl into the solution at 0°C to quench the reaction, extract the solution with 30 mL of ethyl acetate for 3 times, dry the combined organic phases by using anhydrous Na2SO4, filter to remove impurities, remove the solvent by reducing pressure, and obtain a crude product; purify the crude product by column chromatography, wherein the eluent is prepared by using petroleum ether and ethyl acetate in a ratio of 10:1; obtain 2.2 g of yellow oily product (R)-4, that is, 2'-hydroxy-2-(methoxymethoxy)-[1,1'-binaphthalen]-3-carbaldehyde, with a yield of 55%, and its 1 H NMR is shown in Figure 4 , 13 C NMR is shown in Figure 5 ;

[0048] S5, (R)-4 (1.68 mmol, 0.6 g) and potassium carbonate (3.36 mmol, 348 mg) were dissolved in 30 mL of acetonitrile, and after stirring at 50°C for 3 h, 4-(bromomethyl)benzeneboronic acid (2.52 mmol, 541.8 mg) was added and the reaction was allowed to proceed for 21 h;

[0049] S6, After the reaction was completed, the residue was washed with acetonitrile, and for the unreacted anhydrous potassium carbonate found, the acetonitrile was suspended and extracted with 30 mL of ethyl acetate three times, and the organic phase was combined and dried over anhydrous Na2SO4, and filtered to remove impurities; then the solvent was removed by reduced pressure to obtain the crude product; the crude product was purified by column chromatography, and the eluent was prepared using petroleum ether and ethyl acetate in a ratio of 30:1 to obtain 468 mg of yellow product (R)-2, i.e., (4-((((3'-formyl-2'-(methoxymethoxy)-[1,1'-binaphthalenyl]-2-yl)oxy)methyl)phenyl), with a yield of 57%, and its 1 H NMR is shown as follows: Figure 6 , 13 CNMR is shown as follows: Figure 7 ;

[0050] S7, (R)-2 (0.95 mmol, 468 mg) was dissolved in dichloromethane and stirred, and 1 mL of HCl was added to the flask;

[0051] S8, The reaction was continuously tracked for 6 h, and the spot plate result had only one product spot, indicating that the reaction was complete, at which time 100 mL of ultrapure water was added to the flask, and NaHCO3 solid was slowly added several times until the bubbles disappeared;

[0052] S9, 20 mL of ethyl acetate was used to extract three times, the organic phase was combined, dried over anhydrous Na2SO4, and filtered to remove impurities, and then the solvent was removed by reduced pressure to obtain 398 mg of yellow product (R)-3, i.e., (4-((((3'-formyl-2'-hydroxy-[1,1'-binaphthalenyl]-2-yl)oxy)methyl)phenyl)boronic acid, with a yield of 93%, and its 1 H NMR is shown as follows: Figure 8 , 13 C NMR is shown as follows: Figure 9 .

[0053] In the present application, the 1 H NMR is shown as follows: 1H NMR (400 MHz, Chloroform-d) δ 8.03 (d, J = 9.1 Hz, 1H), 7.91 (d, J = 8.6 Hz, 2H), 7.87 (d, J = 8.1 Hz, 1H), 7.60 (d, J = 9.1 Hz, 1H), 7.44 - 7.38 (m, 1H), 7.36 (d, J = 8.9 Hz, 1H), 7.31 (s, 1H), 7.30 (s, 1H), 7.23 (s, 1H), 7.21 (d, J = 8.1 Hz, 1H), 7.08 (d, J = 8.4 Hz, 1H), 5.13 - 5.03 (m, 2H), 3.18 (s, 3H).

[0054] Example 2

[0055] A method for preparing a fluorescent probe for recognizing chiral glucose, comprising the following steps:

[0056] In Example 1, the raw material (R)-6 in step S1 is replaced by (S)-6, and the remaining steps are the same as those in Example 1 to synthesize (S)-3. 1 The H NMR diagram is shown in Figure 10 .

[0057] A solution configuration for fluorescence testing of the (S)-3 probe, comprising the following steps:

[0058] P1, preparing a (S)-3 mother liquor: an appropriate amount of (S)-3 is weighed using an accurate analytical balance, and after being dissolved in DMSO, a standard solution with a concentration of 0.8 mM is prepared in a centrifuge tube.

[0059] P2, preparing an amino sugar solution: different amounts of amino sugar are weighed according to the concentration relationship between the amino sugar and (S)-3, and after being dissolved in ultrapure water, a 6.4 mM amino sugar solution is prepared.

[0060] P3, preparing a zinc acetate solution: according to the molar relationship between (S)-3 and Zn 2+ , an ultrapure water is prepared to prepare a 1.6 mM zinc acetate solution.

[0061] (S)-3 probe fluorescence testing method:

[0062] P1, first use a clean centrifuge tube to load 50 μL of (S)-3 mother liquor;

[0063] P2, add 250 μL of methanol, and at the same time, quickly add 50 μL of amino sugar solution;

[0064] P3, to adjust the volume of the reaction solution, immediately add 50 μL zinc acetate solution using the same pipette, so that the total volume reaches 400 μL;

[0065] P4, after 3 h of reaction, add 3.6 mL of methanol to the centrifuge tube for fluorescence test, and the results are shown in Figure 12 and Figure 13 .

[0066] In the methanol solution, the probe (S)-3 can detect two types of glucose, and it can be known from Figure 12 that L-glucose does not react with the probe, while D-glucose reacts with the probe (S)-3, achieving the effect of fluorescence enhancement.

[0067] In the methanol solution, the probe (S)-3 can detect 17 kinds of amino sugars, and it can be known from Figure 13 that the probe (S)-3 has good recognition effect on D-glucose.

[0068] Finally, it should be pointed out that the above preferred embodiments are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present application.

Claims

1. A method for preparing a fluorescent probe for recognizing D-glucose, characterized in that: Includes the following steps, S1. Weigh (R)-6 and dissolve it completely in ultra-dry DCM. After cooling the solution to 0°C, add DIPEA dropwise and react for 3 h. Then slowly add bromomethyl methyl ether dropwise and react for 1 h. The (R)-6 is (R)-BINOL. S2. The reaction was quenched by adding 2M HCl at 0℃, followed by extraction with ethyl acetate and combining the organic phases. The mixture was then dried and filtered to remove impurities. The solvent was removed by vacuum to obtain the crude product. The crude product was purified by column chromatography to obtain a white solid product (R)-5, which is 2'-(methoxymethoxy)-[1,1'-binaphthyl]-2-ol. S3. Dissolve (R)-5 completely in ultra-dry THF. After cooling the solution to -78°C, slowly add n-butyllithium dropwise and allow it to return to room temperature for 2 hours. Then, cool the solution to 0°C again and slowly add DMF dropwise and allow it to return to room temperature for 1 hour. S4. The reaction was quenched by adding saturated NH4Cl at 0℃ and extracted with ethyl acetate. The organic phases were combined, dried, and filtered to remove impurities. The solvent was then removed by vacuum to obtain a crude product. The crude product was purified by column chromatography to obtain a yellow oily product (R)-4, wherein (R)-4 is 2'-hydroxy-2-(methoxymethoxy)-[1,1'-binaphthyl]-3-carboxaldehyde. S5. Dissolve (R)-4 and potassium carbonate completely in 30 mL of acetonitrile, heat and stir at 50 °C for 3 h, then add 4-(bromomethyl)phenylboronic acid and react for 21 h. S6. After the reaction is complete, the residue is washed with acetonitrile, extracted with ethyl acetate, the organic phases are combined, dried, and filtered to remove impurities; the solvent is then removed under reduced pressure to obtain the crude product; the crude product is purified by column chromatography to obtain the yellow product (R)-2, wherein (R)-2 is (4-(((3'-formyl-2'-(methoxymethoxy)-[1,1'-binaphthyl]-2-yl)oxy)methyl)phenyl). S7. Dissolve (R)-2 completely in dichloromethane and stir well. Then add 1 ml of HCl to the flask. The (R)-2 is 0.95 mmol, 468 mg. S8. Continue to monitor the reaction until only one product spot is found on the TLC plate. At this point, add ultrapure water to the flask, and then add NaHCO3 solid multiple times until the bubbles disappear. S9. Extracted three times with ethyl acetate, the organic phases were combined and dried, and filtered to remove impurities. The solvent was then removed under reduced pressure to finally obtain the yellow product (R)-3, which is (4-(((3'-formyl-2'-hydroxy-[1,1'-binaphthyl]-2-yl)oxy)methyl)phenyl)boronic acid.

2. The method for preparing a fluorescent probe for recognizing D-glucose according to claim 1, characterized in that: In steps S2, S4, S6, and S9, anhydrous Na2SO4 is used for the drying process.

3. The method for preparing a fluorescent probe for recognizing D-glucose according to claim 1, characterized in that: In step S2, when purifying the crude product by column chromatography, the eluent is prepared using a ratio of petroleum ether to ethyl acetate of 40:1; in step S4, when purifying the crude product by column chromatography, the eluent is prepared using a ratio of petroleum ether to ethyl acetate of 10:1; in step S6, when purifying the crude product by column chromatography, the eluent is prepared using a ratio of petroleum ether to ethyl acetate of 30:1.

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