[2.2] cyclic photochromic coumarin fluorescent probes, methods of making and methods of detecting chiral molecules

Abstract

The application discloses a [2.2] cyclophane coumarin fluorescent probe, a preparation method and a method for detecting chiral molecules, and the fluorescent probe comprises a fluorescent probe structure, and the fluorescent probe structure is shown as formula (I). The [2.2] cyclophane skeleton is introduced into the coumarin molecule, and the fluorescent probe can be applied to fluorescence recognition of chiral molecules. The fluorescent probe can chemoselectively and enantioselectively recognize histidine in a mixed solution of tetrahydrofuran and HEPES aqueous solution, and can quantitatively detect the concentration of histidine and the ee value of histidine. The probe is an achiral fluorescent probe, can reduce the difficulty of separation of chiral compounds, greatly reduces the synthesis cost of the fluorescent probe, and opens up a new amino acid recognition probe.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probe technology, specifically relating to a [2.2] cyclocoumarin fluorescent probe, its preparation method, and a method for detecting chiral molecules. Background Technology

[0002] Chirality is a fundamental property of nature; chiral compounds are mirror images of each other but cannot overlap. Chiral α-amino acids are important organic molecules that play an indispensable role in the biological, medical, and chemical industries, serving as crucial starting materials and chiral sources for various chiral drugs and functional organic molecules. D-amino acids exist in organisms and perform specific physiological functions in mammals and humans, while L-amino acids are widely distributed in nature, such as in proteins and peptides. Due to the importance and ease of preparation of chiral amino acids, many rapid and effective chiral identification methods have been reported for determining the concentration or enantiomeric composition of amino acids. Among these methods, fluorescent probe recognition has attracted widespread attention for determining the concentration and enantiomeric composition of chiral amino acids due to its advantages such as fast response, high sensitivity, non-invasive real-time imaging, and high-throughput analysis. However, most of these fluorescent probes are chiral, including axial chirality and central chirality. Most of them cannot chemically selectively recognize one of the 18 common amino acids and cannot quantitatively detect amino acid concentration and ee value [ee=([L]-[D]) / ([L]+[D])]. Summary of the Invention

[0003] The purpose of this invention is to provide a [2.2] cyclocoumarin fluorescent probe, its preparation method, and a method for detecting chiral molecules, in order to solve the problem that fluorescent probes cannot chemically selectively recognize amino acids and cannot quantitatively detect amino acids.

[0004] In a first aspect, embodiments of the present invention provide a [2.2] cyclocoumarin fluorescent probe, comprising: a fluorescent probe structure, the fluorescent probe structure being as shown in formula (Ⅰ):

[0005]

[0006] Where R is Br or H.

[0007] Secondly, embodiments of the present invention provide a method for preparing a [2.2] cyclocoumarin fluorescent probe, comprising:

[0008] Cycloform compound and 4-bromo-7-(dimethylamino)-coumarin were added to a reaction flask, a catalyst was added, and a solvent was added. The mixture was stirred and reacted to obtain [2.2] cycloform coumarin fluorescent probe; the catalyst included at least one of triethylamine and diisopropylethylamine.

[0009] The cycloform compound includes at least one of 4-amino[2,2]cycloform and 4-amino-12-bromo[2,2]cycloform.

[0010] Optionally, the molar ratio of the cycloform compound, 4-bromo-7-(dimethylamino)-coumarin to the catalyst is (0.5-1.5):(0.5-1.5):(3-6).

[0011] Optionally, a catalyst may be added under a nitrogen atmosphere.

[0012] Optionally, the reaction temperature is 60-90℃.

[0013] Optionally, the reaction time is 16-36 hours.

[0014] Optionally, the solvent includes at least one of ethanol and methanol.

[0015] Optionally, it also includes:

[0016] After stirring the reaction, the reaction product was subjected to vacuum distillation to remove the solvent, then washed with the eluent, and purified by column chromatography to obtain the [2.2] cyclocoumarin fluorescent probe.

[0017] The eluent includes at least one of petroleum ether, ethyl acetate, and dichloromethane.

[0018] Optionally, the eluent comprises petroleum ether and ethyl acetate, wherein the volume ratio of petroleum ether to ethyl acetate is 4:1 to 1:1.

[0019] Thirdly, embodiments of the present invention provide a method for detecting chiral molecules, comprising:

[0020] The cyclocoumarin fluorescent probe, auxiliary solution, buffer solution and histidine described in the above embodiment [2.2] are mixed, the fluorescence response value is measured, and at least one of the configuration, concentration and enantiomer composition ratio of the analyte is determined based on the fluorescence response value.

[0021] The auxiliary agent liquid includes at least one of tetrahydrofuran, N,N-dimethylformamide and dimethyl sulfoxide;

[0022] The buffer solution includes at least one of HEPES buffer and phosphate buffer.

[0023] The [2.2] cyclomimelic coumarin fluorescent probe of this invention, by introducing the [2.2] cyclomimelic backbone into the coumarin molecule, can be applied to the fluorescent recognition of chiral molecules. This fluorescent probe can chemically and enantioselectively recognize histidine in a mixed solution of tetrahydrofuran and HEPES aqueous solution, and can quantitatively detect the concentration and ee value of histidine. As a non-chiral fluorescent probe, it can reduce the difficulty of separating chiral compounds, greatly reduce the synthesis cost of fluorescent probes, and open up new avenues for amino acid recognition probes. Attached Figure Description

[0024] Figure 1a These are the 1H NMR spectra of bromocyclophosphamide coumarin from Example 1;

[0025] Figure 1b These are the carbon spectral data of bromocyclocoumarin in Example 1;

[0026] Figure 1c These are the mass spectrometry data of the bromocyclohexane imitation coumarin in Example 1;

[0027] Figure 2a These are the 1H NMR spectra of cyclocoumarin in Example 2;

[0028] Figure 2b These are the carbon spectral data of cyclocoumarin in Example 2;

[0029] Figure 2c These are the mass spectrometry data of the cyclic imitation tangerine in Example 2;

[0030] Figure 3a The fluorescence response of bromocyclocoumarin to D- and L-histidine in a 1:1 mixture of tetrahydrofuran and HEPES aqueous solution is shown.

[0031] Figure 3b The fluorescence response of cyclocoumarin to D- and L-histidine in a 1:1 mixture of tetrahydrofuran and HEPES aqueous solution is shown.

[0032] Figure 4a The fluorescence intensity of bromocyclocoumarin changes with increasing D- / L-histidine concentration;

[0033] Figure 4b The fluorescence intensity of cyclocoumarin changes with increasing D- / L-histidine concentration;

[0034] Figure 5a The fluorescence intensity of bromocyclocoumarin varies with the enantiomeric composition ratio of histidine.

[0035] Figure 5b The fluorescence intensity of cyclocoumarin varies with the ratio of histidine enantiomers.

[0036] Figure 6a The fluorescence response of bromocyclocoumarin to 18 amino acids is shown.

[0037] Figure 6b The fluorescence response of cyclocoumarin to 18 amino acids is shown. Detailed Implementation

[0038] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] The terms "first," "second," etc., used in the specification and claims of this invention are used to distinguish similar objects and are not used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that embodiments of the invention can be implemented in orders other than those illustrated or described herein. Furthermore, in the specification and claims, "and / or" indicates at least one of the connected objects, and the character " / " generally indicates that the preceding and following objects are in an "or" relationship.

[0040] This invention provides a [2.2] cyclocoumarin fluorescent probe, comprising: a fluorescent probe structure as shown in formula (Ⅰ):

[0041]

[0042] Where R is Br or H.

[0043] The [2.2] cyclomimerocoumarin fluorescent probe of this invention, by introducing the [2.2] cyclomimero backbone into the coumarin molecule, can be applied to the fluorescent recognition of chiral molecules. This fluorescent probe can chemically and enantioselectively recognize histidine in a mixed solution of tetrahydrofuran:HEPES aqueous solution, and can quantitatively detect the concentration and ee value of histidine. As a non-chiral fluorescent probe, it can reduce the difficulty of separating chiral compounds, greatly reduce the synthesis cost of fluorescent probes, and open up new avenues for amino acid recognition probes.

[0044] This invention provides a method for preparing a [2.2] cyclocoumarin fluorescent probe, comprising:

[0045] Cycloform compound and 4-bromo-7-(dimethylamino)-coumarin were added to a reaction flask, a catalyst was added, and a solvent was added. The mixture was stirred and reacted to obtain [2.2] cycloform coumarin fluorescent probe; the catalyst included at least one of triethylamine and diisopropylethylamine.

[0046] The cycloform compound includes at least one of 4-amino[2,2]cycloform and 4-amino-12-bromo[2,2]cycloform.

[0047] The catalyst can be added under an inert environment. Similarly, triethylamine can be added under an inert environment, such as nitrogen, argon, or helium. The solvent can be selected from at least one of ethanol and methanol; for example, ethanol can be used. The reaction can be carried out at 60-90°C, for example, at 78°C.

[0048] Optionally, the molar ratio of the cycloform compound, 4-bromo-7-(dimethylamino)coumarin, and triethylamine can be (0.5-1.5):(0.5-1.5):(3-6).

[0049] Optionally, a catalyst may be added under a nitrogen atmosphere.

[0050] Optionally, the reaction temperature can be 60-90℃, for example, the reaction temperature can be 60, 70, 80 or 90℃.

[0051] Optionally, the reaction time can be 16-36 hours, for example, the reaction time can be 16, 20, 26, 30 or 36 hours.

[0052] Optionally, the solvent may include at least one of ethanol and methanol; for example, the solvent may be selected from ethanol.

[0053] Optionally, the preparation method may further include:

[0054] After stirring the reaction, the reaction product was subjected to vacuum distillation to remove the solvent, then washed with the eluent, and purified by column chromatography to obtain the [2.2] cyclocoumarin fluorescent probe.

[0055] The eluent includes at least one of petroleum ether, ethyl acetate, and dichloromethane. The eluent may include petroleum ether and ethyl acetate. The solvent is removed by vacuum distillation, and the eluent is washed with petroleum ether and ethyl acetate. The eluent is then purified by column chromatography to obtain a high-purity [2.2] cyclocoumarin fluorescent probe.

[0056] Optionally, the eluent comprises petroleum ether and ethyl acetate, wherein the volume ratio of petroleum ether to ethyl acetate is 4:1 to 1:1, for example, the volume ratio of petroleum ether to ethyl acetate can be 3:1 or 1:1.

[0057] This invention provides a method for detecting chiral molecules, comprising:

[0058] The cyclocoumarin fluorescent probe, auxiliary solution, buffer solution and histidine described in the above embodiment [2.2] are mixed, the fluorescence response value is measured, and at least one of the configuration, concentration and enantiomer composition ratio of the analyte is determined based on the fluorescence response value.

[0059] The auxiliary agent liquid includes at least one of tetrahydrofuran, N,N-dimethylformamide and dimethyl sulfoxide;

[0060] The buffer solution includes at least one of HEPES (4-hydroxyethylpiperazine ethanesulfonic acid) buffer and phosphate buffer. For example, the auxiliary solution can be tetrahydrofuran, and the buffer solution can be HEPES buffer. The auxiliary solution can be tetrahydrofuran, and the buffer solution can be HEPES buffer. The tetrahydrofuran:HEPES solution can be 1:1. This fluorescent probe can achieve enantioselective recognition of histidine in a mixture of tetrahydrofuran and HEPES solution, and can quantitatively detect the concentration and ee value of histidine.

[0061] The fluorescent probe of this invention is synthesized by introducing a [2,2]cycloform group into the coumarin structure. The properties exhibited by the resulting molecule are the result of the combined action of two groups, not a single group. The coumarin group provides a reaction site for amino acid recognition, but it does not selectively recognize amino acids. The introduction of the [2,2]cycloform group increases the steric hindrance of the probe, preventing π-π stacking. Thus, by adding D / L-histidine with different configurations, different chiral enantiomers are formed. These chiral enantiomers and their corresponding excess amino acids form chiral aggregates at different rates in a mixed solution of tetrahydrofuran and HEPES aqueous solution, resulting in different photophysical properties. Furthermore, this probe can specifically recognize histidine from 18 common amino acids in a mixed solution of tetrahydrofuran and HEPES aqueous solution. This invention overcomes the limitation of most fluorescent probes that can only recognize amino acids in organic reagents. It allows for the design and synthesis of fluorescent probes that recognize amino acids using the [2,2]cycloform group, and achieves chiral recognition using only racemic probes, simplifying the synthesis steps of fluorescent probes and opening up new avenues for amino acid recognition.

[0062] The fluorescent probes of this invention exhibit good stability, as well as high chemoselectivity and enantioselectivity. Racemic bromocyclocoumarin and cyclocoumarin can be used as chiral fluorescent sensors to recognize D / L-histidine in a mixed solution of tetrahydrofuran and HEPES aqueous solution, showing a fluorescence response to D-histidine exceeding that to L-histidine, with an ef value (ef=(I D -I0) / (I L -I0),I D The fluorescence intensity of bromocyclocoumarin / cyclocoumarin plus D-histidine, I LThe fluorescence intensity of bromocyclocoumarin / cyclocomarin plus L-histidine (I0 is the fluorescence intensity of bromocyclocomarin / cyclocomarin) is 17.3 and 8.7, respectively. Bromocyclocomarin and cyclocomarin can also be used to quantitatively determine the concentration and enantiomeric composition of D / L-histidine. This probe exhibits high chemoselectivity and enantioselectivity fluorescence recognition only for D / L-histidine among 18 pairs of common amino acid enantiomeric pairs.

[0063] The present invention will be further illustrated below through some specific embodiments.

[0064] The reaction formula is as follows:

[0065]

[0066] When R is Br, bromocyclocoumarin is prepared; when R is H, cyclocoumarin is prepared.

[0067] Example 1

[0068] [2.2] The preparation method of the cyclocoumarin fluorescent probe is as follows:

[0069] Under nitrogen atmosphere, 4-bromo-7-(dimethylamino)coumarin (121 mg, 0.41 mmol), 4-amino-12-bromo[2.2]cycloform (125 mg, 0.41 mmol), and triethylamine (207 mg, 2.05 mmol) were added to 10 mL of ethanol and stirred at 78 °C for 24 hours. After the reaction was complete, the reaction solution was cooled to room temperature and distilled under reduced pressure. The resulting mixture was then purified by column chromatography (petroleum ether: ethyl acetate = 2:1) to give the final product, bromocycloform coumarin (90 mg, 38%), as a yellow solid.

[0070] Figure 1a , Figure 1b , Figure 1c These are the 1H NMR, 1C NMR, and mass spectrometry data for bromocyclocoumarin, as detailed below:

[0071] 1H NMR (500MHz, CDCl3) δ12.75(s,1H),10.29(s,1H),7.39(d,J=1.8Hz,1H),6.90(d,J=1.3Hz,1H),6.84(d,J=9.5 Hz,1H),6.64(d,J=1.1Hz,2H),6.57(d,J=7.8Hz,1H),6.52(dd,J=8.0,1.8Hz,1H),6.36(d,J=2.6Hz,1H),6.07( dd,J=9.5,2.7Hz,1H),3.47(ddd,J=13.4,9.8,2.1Hz,1H),3.16–3.10(m,1H),3.09–3.05(m,2H),3.05–3.01(m, 1H),2.98(s,6H),2.97–2.92(m,1H),2.84(ddd,J=13.3,10.3,6.7Hz,1H),2.65(ddd,J=13.2,10.7,6.4Hz,1H). 13 C NMR (126MHz, CDCl3) δ191.7,163.9,157.7,157.6,154.1,142.3,141.8,138.6,138.0,136.0,135.4,13 5.3,132.6,132.4,131.8,128.8,127.0,126.7,108.0,101.3,98.1,95.8,39.8,35.5,33.1,32.6,32.5.

[0072] Example 2

[0073] Under nitrogen atmosphere, 4-bromo-7-(dimethylamino)coumarin (147 mg, 0.5 mmol), 4-amino[2,2]cycloform (112 mg, 0.5 mmol), and triethylamine (347 μL, 2.5 mmol) were added to 10 mL of ethanol and stirred at 78 °C for 24 hours. After the reaction was complete, the reaction solution was cooled to room temperature and distilled under reduced pressure. The resulting mixture was then purified by column chromatography (petroleum ether: ethyl acetate = 3:1) to give the final product, cycloform coumarin (92 mg, 42%), as a yellow solid.

[0074] Figure 2a , Figure 2b , Figure 2c These are the 1H NMR, 1C NMR, and mass spectrometry data for cyclocoumarin, as detailed below:

[0075] 1H NMR (500MHz, CDCl3) δ12.77 (s, 1H), 10.29 (s, 1H), 7.31 (dd, J = 8.1, 2.0Hz, 1H), 6.76 ( d,J=9.5Hz,1H),6.66(dd,J=7.8,1.8Hz,1H),6.58(ddt,J=7.8,4.9,2.5Hz,3H),6.48( dd,J=8.1,2.0Hz,1H),6.35(d,J=2.7Hz,1H),6.18(d,J=1.7Hz,1H),6.04(dd,J=9.5, 2.7Hz,1H),3.10(q,J=3.8Hz,4H),3.09–2.99(m,3H),2.97(s,6H),2.71–2.62(m,1H). 13 C NMR (126MHz, CDCl3) δ191.6,163.9,157.7,157.5,154.0,142.3,139.7,139.1,137.8,135.9,135.9,13 3.6,133.3,132.3,132.1,131.6,128.9,128.3,107.9,101.3,98.1,95.7,39.8,35.2,34.8,34.0,32.6.

[0076] Example 3

[0077] [2.2] Conditions for the recognition of histidine by the cyclocoumarin fluorescent probe

[0078] 1. Different pH values

[0079] Take 5.9575g of HEPES and 7.5g of sodium chloride, make up to 1L, and divide evenly into 12 conical flasks. Add sodium hydroxide solution and hydrochloric acid solution respectively to prepare buffer solutions with pH values ​​of 1.5, 2.5, 3.5, 4.5, 5.5, 6.5, 7.5, 8.5, 9.5, 10.5, 11.5 and 12.5.

[0080] Weigh bromocyclocoumarin / cyclocoumarin into a centrifuge tube and add tetrahydrofuran to prepare a solution with a concentration of 2×10⁻⁶. - 3 Concentrated storage at mol / L;

[0081] Weigh D-histidine and L-histidine into separate glass bottles, and add HEPES buffer of different pH values ​​to prepare solutions with a concentration of 8 × 10⁻⁶. -2 A solution of mol / L;

[0082] Take 300 μL of D-histidine or L-histidine solution and add it to 240 μL of tetrahydrofuran. Then add 60 μL of bromocyclocoumarin / cyclocoumarin concentrate and react at 37 °C for 2 h. Add 1200 μL of HEPES buffer and 1200 μL of tetrahydrofuran at different pH values ​​respectively. The excitation wavelength is 360 nm.

[0083] The results showed that the probe selectively recognized histidine in the range of 2.5 to 11.5, with the best enantioselectivity for histidine at pH 7.5.

[0084] 2. Tetrahydrofuran and HEPES in different proportions

[0085] (1) Preparation of HEPES buffer solution with pH=7.5: Take 5.9575g of HEPES, 495mg of sodium hydroxide and 7.5g of sodium chloride, and make up to 1L.

[0086] (2) Weigh bromocyclocoumarin / cyclocoumarin into a centrifuge tube, add tetrahydrofuran to prepare a solution with a concentration of 2×10⁻⁶. - 3 Concentrated storage at mol / L;

[0087] (3) Weigh D-histidine / L-histidine into glass bottles respectively, and add HEPES buffer solution with pH=7.5 to prepare a solution with a concentration of 8×10⁻⁶. -2 A solution of mol / L;

[0088] (4) Take 60 μL of the concentrated solution and add it to 9 centrifuge tubes. Add 240 μL of tetrahydrofuran and 300 μL of D / L-histidine to each tube. React at 37 °C for 2 h. Then add 2.4, 2.1, 1.8, 1.5, 1.2, 0.9, 0.6, 0.3, and 0 mL of THF solution respectively. Then add HEPES buffer to each centrifuge tube until the total liquid volume is 3 mL, thus obtaining mixed solutions with water contents of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90%. The excitation wavelength is 360 nm.

[0089] During the recognition of histidine by this probe under different ratios of tetrahydrofuran and HEPES, the enantioselectivity was good in the range of 30%-70% HEPES content, but the enantioselectivity for histidine was best at 50% HEPES content.

[0090] Example 4

[0091] Histidine recognition

[0092] 1. Chiral recognition of histidine

[0093] (1) Preparation of HEPES buffer solution with pH=7.5: Take 5.9575g of HEPES, 495mg of sodium hydroxide and 7.5g of sodium chloride, and make up to 1L.

[0094] (2) Weigh bromocyclocoumarin / cyclocoumarin into a centrifuge tube, add tetrahydrofuran to prepare a solution with a concentration of 2×10⁻⁶. - 3 Concentrated storage at mol / L;

[0095] (3) Weigh D-histidine and L-histidine into glass bottles respectively, and add HEPES buffer solution with pH=7.5 to prepare a solution with a concentration of 8×10⁻⁶. -2 A solution of mol / L;

[0096] (4) Take 300 μL of D-histidine or L-histidine solution and add it to 240 μL of tetrahydrofuran, then add 60 μL of bromocyclocoumarin concentrate. React at 37°C for 2 h, and finally add 1200 μL of water and 1200 μL of tetrahydrofuran. Take 210 μL of D-histidine or L-histidine solution and add it to 240 μL of tetrahydrofuran, then add 60 μL of bromocyclocoumarin concentrate. React at 37°C for 2 h, and finally add 1200 μL of water and 1200 μL of tetrahydrofuran. The excitation wavelength is 360 nm.

[0097] [2.2] The results of the enantioselective recognition of histidine by the cyclocoumarin / cyclocoumarin fluorescent probe are shown in [the table below]. Figure 3a and Figure 3b As shown in the figure, bromocyclocoumarin / cyclocoumarin exhibit different fluorescence responses to D- and L-histidines, with a higher fluorescence response to D-histidine than to L-histidine, and ef values ​​of 17.3 and 8.7, respectively, indicating good enantioselectivity.

[0098] 2. Quantitative detection of D / L-histidine concentration

[0099] (1) Preparation of HEPES buffer solution with pH=7.5: Take 5.9575g of HEPES, 495mg of sodium hydroxide and 7.5g of sodium chloride, and make up to 1L.

[0100] (2) Weigh bromocyclocoumarin / cyclocoumarin into a centrifuge tube, add tetrahydrofuran to prepare a solution with a concentration of 2×10⁻⁶. - 3 Concentrated storage at mol / L;

[0101] (3) Weigh D-histidine and L-histidine into glass bottles respectively, and add HEPES buffer solution with pH=7.5 to prepare a solution with a concentration of 8×10⁻⁶. -2 A solution of mol / L;

[0102] (4) Take 15, 30, 60, 90, 120, 150, 180, 210, 240, 270, and 300 μL of D-histidine or L-histidine solution into glass bottles, respectively, so that the concentrations of amino acids are 0.4, 0.8, 1.6, 2.4, 3.2, 4.0, 4.8, 5.6, 6.4, 7.2, and 8.0 μm, respectively. Then add 285, 270, 240, 210, 180, 150, 120, 90, 60, 30, and 0 μL of HEPES buffer solution, respectively. Then add 240 μL of tetrahydrofuran to each of the 11 glass bottles, followed by 60 μL of bromocyclocoumarin / cyclocoumarin concentrate. React at 37°C for 2 h. Finally, add 1200 μL of water and 1200 μL of tetrahydrofuran. The excitation wavelength is 360 nm.

[0103] Depend on Figure 4a It was found that bromocyclocoumarin recognizes histidine in the range of 0.4-8 mM, and the concentration of D-histidine has a good linear relationship with fluorescence intensity (R0). 2 =0.99473), and also showed a good linear relationship with L-histidine (R = 0.99473). 2 =0.99904). By Figure 4b It was found that cyclocoumarin recognized histidine in the range of 0.4-5.6 mM, and the concentration of D-histidine showed a good linear relationship with fluorescence intensity (R0). 2 =0.99859), and also showed a good linear relationship with L-histidine (R = 0.99859). 2 =0.99644). Therefore, bromocyclocoumarin / cyclocoumarin can be used to quantitatively detect histidine concentration.

[0104] 3. Quantitative detection of D / L-histidine ee%

[0105] (1) Preparation of HEPES buffer solution with pH=7.5: Take 5.9575g of HEPES, 495mg of sodium hydroxide and 7.5g of sodium chloride, and make up to 1L.

[0106] (2) Weigh bromocyclocoumarin / cyclocoumarin into a centrifuge tube, add tetrahydrofuran to prepare a solution with a concentration of 2×10⁻⁶. - 3 Concentrated storage at mol / L;

[0107] (3) Weigh D-histidine and L-histidine into glass bottles respectively, and add HEPES buffer solution with pH=7.5 to prepare a solution with a concentration of 4×10⁻⁶. -2 A solution of mol / L;

[0108] (4) Weigh different volumes of D-histidine and L-histidine into glass bottles, add 240 μL of tetrahydrofuran, then add 60 μL of bromocyclocoumarin / cyclocoumarin concentrate, react at 37 °C for 2 h, and finally add 1200 μL of water and 1200 μL of tetrahydrofuran. The ee values ​​and volume ratios of D- and L-histidine are as follows: ee% = 0% (0 μL of D-histidine and 300 μL of L-histidine); ee% = 10% (30 μL of D-histidine and 270 μL of L-histidine); ee% = 20% (60 μL of D-histidine and 240 μL of L-histidine); ee% = 30% (90 μL of D-histidine and 210 μL of L-histidine); ee% = 40% (120 μL of D-histidine and 180 μL of L-histidine); ee% = 50% (150 μL of D-histidine and 270 μL of L-histidine); ee% = 50% (150 μL of D-histidine and 270 μL of L-histidine); ee% = 50% (150 μL of D-histidine and 270 μL of L-histidine); ee% = 10% (30 μL of D-histidine and 270 μL of L-histidine); ee% = 20% (60 μL of D-histidine and 240 μL of L-histidine); ee% = 30% (90 μL of D-histidine and 210 μL of L-histidine); ee% = 40% (120 μL of D-histidine and 180 μL of L-histidine); ee% = 50% (150 μL of D-histidine and 27 ... The excitation wavelength was 360 nm. The excitation wavelengths were: 180 μL of D-histidine and 120 μL of L-histidine; ...

[0109] Depend on Figure 5a and Figure 5b It can be seen that the fluorescence intensity of bromocyclocoumarin / cyclocoumarin at 445 nm / 440 nm has a good linear relationship with the ee value (0-100%), R 2 The values ​​are 0.99013 and 0.99552, respectively. Therefore, this probe can be used to quantitatively detect the ee value of histidine.

[0110] 4. Chemoselective recognition of histidine

[0111] (1) Preparation of HEPES buffer solution with pH=7.5: Take 5.9575g of HEPES, 495mg of sodium hydroxide and 7.5g of sodium chloride, and make up to 1L.

[0112] (2) Weigh bromocyclocoumarin / cyclocoumarin into a centrifuge tube, add tetrahydrofuran to prepare a solution with a concentration of 2×10⁻⁶. - 3 Concentrated storage at mol / L;

[0113] (3) Weigh the enantiomers of histidine (His), phenylalanine (Phe), valine (Val), lysine (Lys), tryptophan (Trp), alanine (Ala), leucine (Leu), proline (Pro), tyrosine (Tyr), serine (Ser), threonine (Thr), cysteine ​​(Cys), methionine (Met), asparagine (Asn), glutamine (Gln), arginine (Arg), aspartic acid (Asp), and glutamic acid (Glu) into glass bottles, and add HEPES buffer at pH 7.5 to prepare solutions with a concentration of 8 × 10⁻⁶. -2 A solution of mol / L;

[0114] (4) Take 300 μL of each of the 18 common amino acid enantiomers in a glass bottle, add 240 μL of tetrahydrofuran, then add 60 μL of bromocyclocoumarin / cyclocoumarin concentrate, react at 37 °C for 2 h, and finally add 1200 μL of water and 1200 μL of tetrahydrofuran. The excitation wavelength is 360 nm.

[0115] Bromocyclocoumarin / cyclocoumarin exhibits highly chemoselective fluorescent recognition of D-histidine in the enantiomeric forms of 18 common amino acids. See details... Figure 6a and Figure 6b .

[0116] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of the present invention.

Claims

1. A [2.2] cyclic coumarin fluorescent probe, characterized in that, include: The fluorescent probe structure is shown in formula (Ⅰ): Where R is Br or H.

2. A method for preparing a [2.2] cyclocoumarin fluorescent probe, characterized in that, include: Cycloform compound and 4-bromo-7-(dimethylamino)-coumarin were added to a reaction flask, a catalyst was added, and a solvent was added. The mixture was stirred and reacted to obtain [2.2] cycloform coumarin fluorescent probe; the catalyst included at least one of triethylamine and diisopropylethylamine. The cycloform compound includes at least one of 4-amino[2,2]cycloform and 4-amino-12-bromo[2,2]cycloform.

3. The preparation method according to claim 2, characterized in that, The molar ratio of the cycloamorph compound, 4-bromo-7-(dimethylamino)-coumarin, and the catalyst was (0.5-1.5):(0.5-1.5):(3-6).

4. The preparation method according to claim 2, characterized in that, Add the catalyst under nitrogen atmosphere.

5. The preparation method according to claim 2, characterized in that, The reaction temperature is 60-90℃.

6. The preparation method according to claim 2, characterized in that, The reaction time is 16-36 hours.

7. The preparation method according to claim 2, characterized in that, The solvent includes at least one of ethanol and methanol.

8. The preparation method according to claim 2, characterized in that, Also includes: After stirring the reaction, the reaction product was subjected to vacuum distillation to remove the solvent, then washed with the eluent, and purified by column chromatography to obtain the [2.2] cyclocoumarin fluorescent probe. The eluent includes at least one of petroleum ether, ethyl acetate, and dichloromethane.

9. The preparation method according to claim 8, characterized in that, The eluent comprises petroleum ether and ethyl acetate, with a volume ratio of petroleum ether to ethyl acetate of 4:1 to 1:

1.

10. A method for detecting chiral molecules, characterized in that, include: Mix the [2.2] cyclocoumarin fluorescent probe, auxiliary solution, buffer solution and histidine as described in claim 1, measure the fluorescence response value, and determine at least one of the configuration, concentration and enantiomer composition ratio of the analyte based on the fluorescence response value; The auxiliary agent liquid includes at least one of tetrahydrofuran, N,N-dimethylformamide and dimethyl sulfoxide; The buffer solution includes at least one of HEPES buffer and phosphate buffer.

Images