Compounds containing a benzothiazole group, fluorescent probes and methods of preparation and use thereof

Abstract

This invention discloses compounds containing benzothiazole groups, fluorescent probes, their preparation methods, and applications. The preparation method of the compounds containing benzothiazole groups includes the following steps: under inert gas protection, (S)-1 and 2-aminobenzothiazoles are added to a mixed solvent of alcohol and dichloromethane, and the reaction is carried out at 50–100°C. After the reaction, the reaction solution is filtered to obtain a filter cake. The filter cake is washed and dried, and then purified by pulping or column chromatography to obtain the product. This invention utilizes a simple amine-aldehyde condensation reaction to obtain a novel Schiff base-type chiral fluorescent probe. The probe is characterized by the incorporation of the benzothiazole fluorophore into the chiral backbone via a -C=N-linking group. The -C=N-linking group increases the degree of conjugation in the molecule, thereby improving the fluorescence quantum yield and fluorescence intensity. This probe achieves chiral fluorescent recognition of various amino acids in a variety of organic solvents, demonstrating universality in amino acid recognition.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probes, and particularly relates to compounds containing benzothiazole groups, fluorescent probes, their preparation methods and applications. Background Technology

[0002] Chiral compounds are widely distributed in nature. As essential biological macromolecules for life activities, such as proteins, polysaccharides, nucleic acids, and enzymes, almost all possess chirality. Chirality also has extensive applications in the chemical and pharmaceutical fields; chiral drugs account for more than half of the new drugs currently under development globally. Chiral amino acids, as one type of chiral compound, have wide applications in life activities, asymmetric synthesis, pharmaceuticals, and food. Obtaining single chiral compounds is crucial. Therefore, various strategies such as chiral resolution and precise chiral synthesis have emerged. Research progress in this field relies heavily on efficient methods for the recognition and detection of chiral molecules.

[0003] Fluorescent probe molecular recognition technology is widely used in the biological and medical fields due to its advantages such as simple operation, high sensitivity, real-time imaging, and readily available instruments. Therefore, developing a fluorescent probe with high enantioselectivity for amino acids and the ability to determine their enantiomer composition is a research endeavor with significant application value.

[0004] Benzothiazoles and their derivatives are a class of very important heterocyclic compounds, widely found in nature and mainly used in medicine, agriculture, and industry. Furthermore, due to their excellent fluorescence quantum yield, benzothiazoles have been extensively incorporated into the molecular structures of fluorescent probes. Existing technology discloses a chiral fluorescent probe containing a benzothiazole group (application number 202210665107.2), which exhibits enantioselective fluorescence enhancement for 6 of the 19 common chiral amino acids (alanine, valine, histidine, threonine, arginine, and asparagine). However, the conjugation degree and enantioselectivity of the probe described in this patent need improvement, and its applicability to chiral substrates needs to be expanded. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a compound containing a benzothiazole group, a fluorescent probe, its preparation method and application.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A compound containing a benzothiazole group has the following chemical structural formula:

[0008]

[0009] The preparation method of the above-mentioned compound containing the benzothiazole group includes the following steps: under the protection of an inert gas, (S)-2,2'-dihydroxy-1,1'-binaphthyl-3,3'-dicarboxaldehyde (i.e., (S)-1) and 2-aminobenzothiazole are added to a mixed solvent of alcohol and dichloromethane, and the reaction is carried out at 50-100°C. After the reaction is completed, the reaction solution is filtered to obtain a filter cake. The filter cake is washed and dried, and then purified by pulping or column chromatography to obtain the compound containing the benzothiazole group.

[0010] (S)-2,2'-dihydroxy-1,1'-binaphthyl-3,3'-dicarboxaldehyde was prepared according to the following literature: Chemical Science, 2014, 5, 3457-3462.

[0011] Preferably, the equivalent (molar) ratio of (S)-2,2'-dihydroxy-1,1'-binaphthyl-3,3'-dicarboxaldehyde and 2-aminobenzothiazole is 0.8-1:1-1.3.

[0012] Preferably, the reaction time is 4 to 24 hours.

[0013] Preferably, the alcohol and dichloromethane are mixed solvents in a volume ratio of 2:1 or 3:1.

[0014] Preferably, the alcohol is methanol or ethanol.

[0015] Preferably, the solvent used for washing is at least one selected from ethanol, methanol, isopropanol, n-butanol, and n-hexane.

[0016] Preferably, the pulping method is as follows: ethyl acetate and petroleum ether are added to the compound containing the benzothiazole group, and the mixture is pulped and purified, wherein the volume ratio of petroleum ether to ethyl acetate is 12:1; and the amount ratio of the compound containing the benzothiazole group to petroleum ether is 60 mg: 36 mL.

[0017] Preferably, the column chromatography method is as follows: the compound containing the benzothiazole group is dissolved in ethyl acetate, and then 200-300 mesh silica gel powder is added and stirred in a rotary dryer before chromatography is performed. A silica gel column is used, the column is alkalized with 0.5% triethylamine, and a mixture of PE (petroleum ether):EA (ethyl acetate) = 10:1 (v / v) is used as the eluent.

[0018] A fluorescent probe having the structure of the compound containing the benzothiazole group.

[0019] Application of the fluorescent probe in the enantioselective fluorescent recognition of chiral amino acids.

[0020] Preferably, the application includes the following method: mixing a fluorescent probe, metal ions, amino acids, and an organic solvent to form a detection system, measuring the fluorescence response of the system, and determining the configuration of the amino acids accordingly.

[0021] Preferably, the amino acid is one of arginine, lysine, phenylalanine, isoleucine, valine, tryptophan, serine, asparagine, histidine, leucine, glutamic acid, and threonine.

[0022] Preferably, the equivalent ratio (molar ratio) of the fluorescent probe, metal ions and amino acids is 1:1 to 9:1 to 50.

[0023] Preferably, the organic solvent is at least one of dimethyl sulfoxide and ethanol.

[0024] Preferably, the metal ion is a zinc ion. The zinc ion is introduced by zinc acetate.

[0025] Preferably, the amino acid is prepared as an amino acid solution with a pH of 6 to 8.8. Specifically, it is prepared using AcOH / AcONa buffer solution with a pH of 6, HEPES buffer solution with a pH of 7.4 and 8.2, and BICINE buffer solution with a pH of 8.8, respectively.

[0026] This invention utilizes a -C=N- linker generated by a simple amine-aldehyde condensation reaction to conjugate a benzothiazole group onto a chiral backbone, resulting in a novel Schiff base-type chiral fluorescent probe. The position of the benzothiazole group on the chiral backbone differs from that of probes previously developed by the applicant (application number 202210665107.2), and the -C=N- linker increases the conjugation degree of the probe molecule, thereby improving the fluorescence quantum yield and enhancing fluorescence. Simultaneously, the N atom on the -C=N- linker also enhances the complexation with metal ions involved in the chiral recognition of amino acids, thereby increasing the rigidity of the molecular structure and achieving improved enantioselectivity for amino acids.

[0027] Compared with the prior art, the beneficial effects of the present invention include:

[0028] The chiral fluorescent probe prepared by this invention can achieve fluorescent recognition of a variety of chiral amino acids in a variety of organic solvents, exhibiting universality in amino acid recognition and extremely high enantioselectivity for arginine. Furthermore, the preparation process of this chiral fluorescent probe is simple and the raw materials are readily available, making it a promising candidate for applications in the fields of fluorescent probes and bioimaging. Attached Figure Description

[0029] Figure 1 The process flow diagram is shown for the preparation of the chiral fluorescent probe (S)-2 according to the present invention.

[0030] Figure 2The chiral fluorescent probe (S)-2 prepared in this invention 1 H-NMR spectrum.

[0031] Figure 3 The chiral fluorescent probe (S)-2 prepared in this invention 13 C-NMR spectrum.

[0032] Figure 4 High-resolution mass spectrometry of the chiral fluorescent probe (S)-2 prepared in this invention.

[0033] Figure 5 The fluorescence spectrum of the chiral fluorescent probe (S)-2 provided by this invention for arginine in dimethyl sulfoxide.

[0034] Figure 6 The fluorescence spectrum of the chiral fluorescent probe (S)-2 provided by this invention for arginine in ethanol.

[0035] Figure 7 The fluorescence spectrum of the chiral fluorescent probe (S)-2 provided by this invention for lysine in ethanol.

[0036] Figure 8 The fluorescence spectrum of the chiral fluorescent probe (S)-2 provided by this invention for phenylalanine in ethanol.

[0037] Figure 9 The fluorescence spectrum of the chiral fluorescent probe (S)-2 provided by this invention for isoleucine in ethanol.

[0038] Figure 10 The fluorescence spectrum of the chiral fluorescent probe (S)-2 provided by this invention for valine in ethanol.

[0039] Figure 11 The fluorescence spectrum of the chiral fluorescent probe (S)-2 provided by this invention for tryptophan in ethanol.

[0040] Figure 12 The fluorescence spectrum of the chiral fluorescent probe (S)-2 provided by this invention for serine in ethanol.

[0041] Figure 13 The fluorescence spectrum of the chiral fluorescent probe (S)-2 provided by this invention against asparagine in ethanol.

[0042] Figure 14 The fluorescence spectrum of the chiral fluorescent probe (S)-2 provided by this invention for histidine in ethanol.

[0043] Figure 15 The fluorescence spectrum of the chiral fluorescent probe (S)-2 provided by this invention for leucine in ethanol.

[0044] Figure 16 The fluorescence spectrum of the chiral fluorescent probe (S)-2 provided by this invention for glutamic acid in ethanol.

[0045] Figure 17 The fluorescence spectrum of the chiral fluorescent probe (S)-2 provided by this invention for threonine in ethanol. Detailed Implementation

[0046] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0047] Example 1

[0048] A method for preparing a compound containing a benzothiazole group, comprising the following steps:

[0049] See Figure 1 , Figure 1 This is a process flow diagram for the chiral fluorescent probe (S)-2.

[0050] Under inert gas protection, (S)-1 and 2-aminobenzothiazole were dissolved in a mixed solvent of ethanol and dichloromethane (volume ratio 2:1) at a 1:1 equivalent ratio. The mixture was heated to 65°C and refluxed. After reacting for 10 hours, heating was stopped. After the reaction solution cooled to room temperature, the reaction solution was filtered. The filter cake was washed with cold anhydrous ethanol and dried to obtain a yellow solid powder, which is the crude product of the compound containing the benzothiazole group.

[0051] The probe product was obtained by purifying 60 mg of the crude compound containing the benzothiazole group by mixing it with 3 mL of ethyl acetate and 36 mL of petroleum ether.

[0052] The structure of the probe was analyzed, see details below. Figures 2-4 Its characterization data are as follows:

[0053] 1 H NMR(400MHz,DMSO-d6)δ11.61(s,1H),10.38(s,1H),9.74(s,1H),8.82(s,1H ),8.65(s,1H),8.13(d,J=8.0Hz,2H),8.01(d,J=8.0Hz,1H),7.65(d,J=7.9H z,1H),7.57(t,J=7.7Hz,1H),7.50–7.45(m,2H),7.43(s,2H),7.33(d,J=8.0 Hz,1H),7.20(t,J=7.6Hz,1H),7.10(d,J=5.3Hz,1H),7.01(d,J=7.4Hz,1H). 13C NMR (400MHz, CDCl3) δ196.87,153.83,138.62,137.63,130.83,130.15,127.83,126.23,1 25.05,124.66,124.37,122.64,122.32,121.09,119.54,116.71.HRMS(ESI):Calculated for C 29 H 19 N₂O₃S([M+H)) + ): m / z 475.1116, found: 475.1118.

[0054] Example 2

[0055] A method for preparing a compound containing a benzothiazole group, comprising the following steps:

[0056] Under inert gas protection, (S)-1 and 2-aminobenzothiazole were dissolved in a mixed solvent of methanol and dichloromethane (volume ratio 3:1) at a 1:1 equivalent ratio. The mixture was heated to 50°C and refluxed. After reacting for 10 hours, heating was stopped. After the reaction solution cooled to room temperature, the reaction solution was filtered, the filter cake was washed with isopropanol, and dried to obtain a solid powder, which is the crude product of the compound containing the benzothiazole group.

[0057] The crude product containing the benzothiazole group was dissolved in ethyl acetate, and then 200-300 mesh silica gel powder was added, mixed, and evaporated to dryness before chromatography. A silica gel column was used, and the column was alkalized with 0.5% triethylamine. After elution with PE:EA = 10:1 (v / v), the eluent containing the benzothiazole compound was collected, evaporated to dryness, and the probe product was obtained.

[0058] Example 3

[0059] The chiral fluorescent probe prepared in Example 1 was dissolved in dimethyl sulfoxide (DMSO) to obtain a 2 mmol / L probe stock solution. Separately, a 6 mmol / L zinc acetate aqueous solution and a 50 mmol / L arginine stock solution (prepared with HEPES buffer at pH 7.4) were prepared. 50 μL of each solution was taken and diluted to 300 μL with 150 μL of DMSO. After reacting for 80 minutes, the solution was diluted again to 3 mL and allowed to stand for 30 minutes before fluorescence testing. The probe concentration in the system was 33.3 μmol / L, with a dimethyl sulfoxide to water volume ratio of 29:1. The test results are as follows: Figure 5 As shown, the fluorescence intensity of the probe differs significantly for different configurations of arginine, with the fluorescence intensity for L-arginine being significantly higher than that for D-arginine. The probe exhibits extremely high enantioselectivity for arginine, with an ef value of approximately 281 at 515 nm.

[0060] Example 4

[0061] Based on Example 3, only the following changes were made: the chiral fluorescent probe prepared in Example 1 was dissolved in ethanol to obtain a 1 mmol / L probe stock solution, and a 6 mmol / L zinc acetate aqueous solution and a 50 mmol / L arginine stock solution were prepared separately.

[0062] After preparing the test samples according to the equivalent ratio described in Example 3, the fluorescence difference was tested. The specific operating steps were the same as in Example 3. The test results are as follows: Figure 6 As shown, the probe also exhibits good enantioselective recognition of arginine in ethanol.

[0063] Example 5

[0064] Based on Example 4, the amino acid was replaced with "lysine," while all other steps remained unchanged. The fluorescence spectrum of the chiral fluorescent probe (S)-2 for lysine in ethanol is as follows. Figure 7 As shown.

[0065] Example 6

[0066] Based on Example 4, the amino acid was replaced with "phenylalanine," while all other steps remained unchanged. The fluorescence spectrum of chiral fluorescent probe (S)-2 in ethanol for phenylalanine is as follows. Figure 8 As shown.

[0067] Example 7

[0068] Based on Example 4, the amino acid was replaced with "isoleucine," while all other steps remained unchanged. The fluorescence spectrum of the chiral fluorescent probe (S)-2 for isoleucine in ethanol is as follows. Figure 9 As shown.

[0069] Example 8

[0070] Based on Example 4, the amino acid was replaced with "valine," while all other steps remained unchanged. The fluorescence spectrum of chiral fluorescent probe (S)-2 for valine in ethanol is as follows. Figure 10 As shown.

[0071] Example 9

[0072] Based on Example 4, the amino acid was replaced with "tryptophan," while all other steps remained unchanged. The fluorescence spectrum of chiral fluorescent probe (S)-2 for tryptophan in ethanol is as follows. Figure 11 As shown.

[0073] Example 10

[0074] Based on Example 4, the amino acid was replaced with "serine," while all other steps remained unchanged. The fluorescence spectrum of the chiral fluorescent probe (S)-2 for serine in ethanol is as follows. Figure 12 As shown.

[0075] Example 11

[0076] Based on Example 4, the amino acid was replaced with "asparagine," while all other steps remained unchanged. The fluorescence spectrum of chiral fluorescent probe (S)-2 for aspartic acid in ethanol is as follows. Figure 13 As shown.

[0077] Example 12

[0078] Based on Example 4, the amino acid was replaced with histidine, while all other steps remained unchanged. The fluorescence spectrum of the chiral fluorescent probe (S)-2 for histidine in ethanol is as follows. Figure 14 As shown.

[0079] Example 13

[0080] Based on Example 4, the amino acid was replaced with leucine, while all other steps remained unchanged. The fluorescence spectrum of chiral fluorescent probe (S)-2 for leucine in ethanol is as follows. Figure 15 As shown.

[0081] Example 14

[0082] Based on Example 4, the amino acid was replaced with "glutamic acid," while all other steps remained unchanged. The fluorescence spectrum of chiral fluorescent probe (S)-2 for glutamic acid in ethanol is as follows. Figure 16 As shown.

[0083] Example 15

[0084] Based on Example 4, the amino acid was replaced with "threonine," while all other steps remained unchanged. The fluorescence spectrum of chiral fluorescent probe (S)-2 for threonine in ethanol is as follows. Figure 17 As shown.

[0085] See Figures 7-17 As can be seen, the probes prepared in this invention have good enantioselective recognition effects on lysine, phenylalanine, isoleucine, valine, tryptophan, serine, asparagine, histidine, leucine, glutamic acid and threonine.

[0086] The specific embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any other corresponding changes and modifications made in accordance with the technical concept of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A compound containing a benzothiazole group, characterized in that, It has the following chemical structural formula:

2. The method for preparing the compound containing the benzothiazole group according to claim 1, characterized in that, The process includes the following steps: under an inert gas atmosphere, (S)-2,2'-dihydroxy-1,1'-binaphthyl-3,3'-dicarboxaldehyde and 2-aminobenzothiazole are added to a mixed solvent of alcohol and dichloromethane, and the reaction is carried out at 50-100°C. After the reaction is completed, the reaction solution is filtered to obtain a filter cake. The filter cake is washed and dried, and then purified by pulping or column chromatography to obtain the compound containing the benzothiazole group.

3. The method for preparing the compound containing a benzothiazole group according to claim 2, characterized in that, The equivalent ratio of (S)-2,2'-dihydroxy-1,1'-binaphthyl-3,3'-dicarboxaldehyde to 2-aminobenzothiazole is 0.8-1:1-1.3; The alcohol and dichloromethane are mixed in a volume ratio of 2:1 or 3:1 to form a solvent; The reaction time is 4 to 24 hours.

4. The method for preparing the compound containing a benzothiazole group according to claim 2 or 3, characterized in that, The alcohol is methanol or ethanol; The solvent used for washing is at least one of ethanol, methanol, isopropanol, n-butanol, and n-hexane.

5. The method for preparing the compound containing a benzothiazole group according to claim 2, characterized in that, The pulping method is as follows: ethyl acetate and petroleum ether are added to the compound containing the benzothiazole group, and the mixture is pulped and purified. The volume ratio of petroleum ether to ethyl acetate is 12:1; the amount ratio of the compound containing the benzothiazole group to petroleum ether is 60 mg: 36 mL. The column chromatography method is as follows: the compound containing the benzothiazole group is dissolved in ethyl acetate, and then 200-300 mesh silica gel powder is added, mixed and evaporated to dryness before chromatography. A silica gel column is used, the column is alkalized with 0.5% triethylamine, and the eluent is petroleum ether:ethyl acetate with a volume ratio of 10:

1.

6. A fluorescent probe, characterized in that, Its structure is that of the compound containing the benzothiazole group as described in claim 1.

7. The application of the fluorescent probe of claim 6 in the enantioselective fluorescent recognition of chiral amino acids.

8. The application according to claim 7, characterized in that, The method includes the following steps: mixing a fluorescent probe, metal ions, amino acids, and an organic solvent to form a detection system, measuring the fluorescence response of the system, and determining the configuration of the amino acids accordingly.

9. The application according to claim 8, characterized in that, The amino acid is one of arginine, lysine, phenylalanine, isoleucine, valine, tryptophan, serine, asparagine, histidine, leucine, glutamic acid, and threonine. The equivalent ratio of the fluorescent probe, metal ions, and amino acids is 1:1 to 9:1 to 50.

10. The application according to claim 8, characterized in that, The organic solvent is at least one of dimethyl sulfoxide and ethanol; The metal ion is a zinc ion; The amino acids are prepared into amino acid solutions with a pH of 6 to 8.8 for use.

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