A benzothiazole organic compound and its application
By using benzothiazole organic compounds to detect the pH value of the solution under a single excitation light, the problems of complex operation and light damage in the existing technology are solved, and high-sensitivity pH detection and RNA detection are achieved, which are used in tumor diagnosis and imaging reagents.
Patent Information
- Application Number
- CN202311471084.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-11-07
- Publication Date
- 2025-09-16
- Estimated Expiration
- 2043-11-07
AI Technical Summary
Existing fluorescent probes require two types of excitation light when detecting the pH value of a solution. The operation is complicated and easily causes fluorescence damage. They have low sensitivity and cannot effectively detect changes in intracellular pH.
A benzothiazole organic compound was designed to detect the pH value of the solution by the fluorescence intensity ratio method under a single excitation light, reducing photodamage, improving sensitivity, and can be used to detect RNA.
It achieves high-sensitivity detection of solution pH under single excitation light, simplifies operation, reduces light damage to fluorescent probes, and can be used to prepare tumor diagnosis and imaging reagents.
Smart Images
Figure CN117510433B_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of chemical medicine, and in particular relates to a benzothiazole organic compound and application thereof. Background Art
[0002] pH value is an important indicator in the field of chemical industry production. It reflects the physical quantity of the acidity and alkalinity of the solution and reflects the hydrogen ions (H + ) concentration. Measuring the pH of a solution can determine its chemical properties and better control and regulate chemical reactions. pH can also reflect changes in the intracellular microenvironment and is closely related to biomolecular interactions, metabolite diffusion, and signal transduction. In laboratory research and industrial production, the pH of a solution is an important quality control parameter. Therefore, detecting pH changes is of great significance to chemistry and biology.
[0003] Currently, the main methods for detecting pH value include pH test paper and pH meter. These methods require a large amount of liquid and direct contact between the solution and the test instrument, which will cause contamination of the measuring instrument and cannot detect pH changes in cells.
[0004] With the development of science and technology, fluorescence has become an important method for detecting the pH of solutions and cells due to its advantages such as high sensitivity and rapid response. Current fluorescent probes generally detect changes in the pH value of solutions by changes in fluorescence intensity, but this method has low detection sensitivity and is easily affected by instrument errors. Ratio-type fluorescent probes can offset the influence of instrument errors by the ratio of fluorescence intensities, thereby improving the accuracy of detection. Current ratio-type fluorescent probes often require two excitation lights when detecting fluorescence intensity, which can easily make the operation complicated. More importantly, increasing the excitation light will cause fluorescence damage to the fluorescent probe and produce fluorescence quenching.
[0005] Therefore, it is necessary to design a fluorescent compound with high pH sensitivity. Under the excitation of a single excitation light, the fluorescence intensity ratio method can be used to reduce the light damage to the organic fluorescent compound and achieve high-sensitivity detection of the pH value of the solution. Summary of the Invention
[0006] Purpose of the Invention: The present invention provides a benzothiazole organic compound and its application, specifically, its application for monitoring solution pH and detecting RNA under single excitation light. Because cellular pH and RNA are associated with various diseases, such as cancer, the multi-purpose organic compound of the present invention can be used to prepare pH-sensitive tumor diagnostic reagents and fluorescent or radioactive tumor imaging reagents.
[0007] Technical solution: The purpose of the present invention is achieved through the following technical solution:
[0008] The present invention provides a benzothiazole organic compound, the chemical name of the compound is: (E)-2-(3-chloro-4-hydroxy-5-methoxyphenyl)-3-methylbenzothiazole-3-iodine salt; its structural formula is:
[0009]
[0010] The present invention also provides an application of the benzothiazole organic compound for observing the pH value of a solution under the excitation of a single excitation light.
[0011] The compound can roughly detect the pH value of a solution under visible light conditions.
[0012] The compound can accurately measure the pH value of a solution under a fluorescence photometer or a fluorescence spectrometer.
[0013] A preferred embodiment of the present invention is to add the compound to the solution to be tested, and detect the fluorescence intensity I of the solution. F , and then calculate the pH value of the solution according to the equation log[(1.5-I) / (I-0.07)]=pH-5.
[0014] The pH value of the solution ranges from 3.0 to 9.0.
[0015] The concentration of the compound in the solution is 5-15 μM. That is, the concentration of the compound in staining cells is 5-15 μM. When measuring the solution at this concentration, there is a lower aggregation quenching effect and a better measurement effect.
[0016] Furthermore, the concentration of the compound in the solution is 10 μM.
[0017] The benzothiazole organic compound of the present invention can be used for detecting the pH value of sewage and the pH value microenvironment of organisms.
[0018] The present invention also provides a method for detecting the pH of a solution using the benzothiazole organic compound, comprising the following steps:
[0019] (1) adding the benzothiazole organic compound to a solution to be tested;
[0020] (2) using a fluorescence intensity detection device to excite the solution under a single excitation light of 470 nm to obtain a fluorescence intensity ratio I of the solution at 530 nm and 590 nm;
[0021] (3) The pH value of the mixed solution is directly calculated according to the equation log[(1.5-I) / (I-0.07)]=pH-5.
[0022] When using existing fluorescent probes to detect the pH value of a solution, the fluorescent compound is typically placed in a cuvette and excited with 405nm light. Spectrum 1 is obtained using a fluorescence spectrometer. The compound is then placed in the instrument and excited with 543nm light to obtain fluorescence spectrum 2 at 543nm. The two spectrum files are opened separately, the fluorescence intensity values in spectrum 1 and spectrum 2 are read, and the ratio of the fluorescence intensity of the fluorescent compound at 550nm in spectrum 1 and 605nm in spectrum 2 is calculated. Finally, the pH value of the solution is calculated based on the obtained fluorescence intensity ratio.
[0023] The detection method provided by the present invention directly uses a single excitation light at 470 nm to generate a corresponding spectrum. The fluorescence intensity within the spectrum is then read to directly calculate the pH value of the solution. The benzothiazole organic compounds described herein can measure the pH value of a solution under a single excitation light, whereas existing detection methods require two separate excitation light beams to measure the pH value. This demonstrates that pH measurement using the compounds described herein is simple to perform and, more importantly, reduces photodamage to the probe due to the reduced number of excitations.
[0024] The inventors further explored the uses of the benzothiazole organic compound and discovered that it can be used to detect RNA. RNA is of great significance in cellular life, playing a crucial role in the transmission of genetic information and protein synthesis. RNA is also closely related to several diseases, making in vitro detection of RNA of great significance.
[0025] Therefore, the present invention also provides the use of the benzothiazole organic compound in detecting RNA.
[0026] The present invention also provides the use of the benzothiazole organic compound in preparing an RNA detection reagent.
[0027] The present invention also provides the use of the benzothiazole organic compound in preparing tumor diagnostic reagents, tumor fluorescence imaging reagents or radioactive imaging reagents.
[0028] The concentration of the compound for staining cells is 1-15 μM. This concentration has low cytotoxicity and good imaging effect.
[0029] Beneficial effects:
[0030] (1) The organic compounds of the present invention impart excellent optical properties by introducing cationic structures such as benzothiazole. By introducing pH-sensitive phenolic hydroxyl groups, the organic compounds exhibit high sensitivity to pH. Due to the introduction of cationic salts, the fluorescence spectrum of the organic compounds red-shifts, resulting in excellent optical properties.
[0031] (2) Furthermore, the benzothiazole organic compounds of the present invention can measure the pH value of a solution under a single excitation light. Therefore, when using the compounds of the present invention to measure pH, the operation is simple. More importantly, due to the reduced number of excitations, photodamage to the probe can be reduced. Therefore, the compounds of the present invention can be used to detect the pH value of sewage and the pH microenvironment of biological organisms.
[0032] (3) Since the pH value and RNA in cells are related to various diseases (cancer, etc.), the organic compounds of the present invention can be used to prepare RNA detection reagents, tumor diagnostic reagents, and tumor fluorescence imaging reagents or radioactive imaging reagents. BRIEF DESCRIPTION OF THE DRAWINGS
[0033] Figure 1 The absorption spectra of TBCL (10 μM) in different solvents.
[0034] Figure 2 The absorption spectra and images of TBCL (10 μM) in solutions with different pH concentrations are shown below. Figure 2 (A) Absorption spectra of TBCL (10 μM) in solutions with different pH concentrations; Figure 2 (B) is a picture of TBCL (10 μM) in solutions with different pH concentrations.
[0035] Figure 3 is the fluorescence spectrum and fitting curve of TBCL (10 μM) in different pH solutions; Figure 3 (A) Fluorescence spectra of TBCL (10 μM) in solutions with different pH concentrations; Figure 3 (B) Fitting curve of TBCL (10 μM) at different pH values.
[0036] Figure 4 is the fluorescence spectrum and fitting curve of TBCL (10 μM) in RNA solutions with different concentrations; Figure 4 (A) Absorption spectra of TBCL (10 μM) in tris buffer with and without RNA; Figure 4 (B) Fluorescence spectra of TBCL (10 μM) in RNA solutions with different concentrations; Figure 4 (C) Fluorescence intensity curve of TBCL (10 μM) in RNA solutions with different concentrations at 570 nm; Figure 4 (D) Fitting curve of TBCL (10 μM) in RNA solutions with different concentrations. DETAILED DESCRIPTION
[0037] The technical solution of the present invention is described in detail below through specific embodiments, but the protection scope of the present invention is not limited to the embodiments.
[0038] The substances in each example are all commercially available products.
[0039] The absorption spectrum was measured using a Hitachi U-2910 spectrophotometer, and the fluorescence spectrum was measured using a Hitachi F-2700 spectrophotometer.
[0040] Example 1 Synthesis of benzothiazole organic compounds (abbreviated as TBCL)
[0041]
[0042] Wherein, the substituent R is an alkyl group. In this embodiment, R is selected from methyl group.
[0043] Benzothiazole iodide (0.291 g, 1 mmol) and 3-chloro-4-hydroxy-5-methoxybenzaldehyde (0.186 g, 1 mmol) were dissolved in 20 mL of methanol and stirred in a flask for 1 hour. After stirring, a few drops of piperidine were added, and the mixture was refluxed at 85°C for 8 hours. After cooling to room temperature, the mixture was washed with petroleum ether. Column chromatography using a mixture of CH2Cl2 and CH3OH (CH2Cl2:CH3OH volume ratio of 10:1 to 6:1) as the eluent yielded 0.16 g of a red solid, chemically designated (E)-2-(3-chloro-4-hydroxy-5-methoxyphenyl)-3-methylbenzothiazole-3-iodide, an organic compound molecule abbreviated as TBCL.
[0044] The structure of the compound was confirmed to be correct by analyzing the compound's hydrogen and carbon spectra. The characterization results are as follows:
[0045] 1 H NMR (400MHz, DMSO-d6) δ (ppm): 8.33 (d, J = 8.0Hz, 1H), 8.11 (d, J = 8.0Hz, 1H), 8.0 (d, J = 16 .0Hz,1H),7.78-7.82(m,2H),7.68-7.72(m,2H),7.55(s,1H),4.26(s,3H),3.92(s,3H).
[0046] 13 C NMR (400MHz, DMSO-d6), δ (ppm): 170.82, 150.03, 148.21, 142.51, 129.45, 127.99, 127 .50,126.83,126.78,124.39,124.27,121.91,116.45,111.33,109.22,56.90,36.26. C 17 H 15 ClINO2S.
[0047] Example 2 Photophysical properties test of organic compound TBCL in different solvents
[0048] Different types of organic solvents (see Figure 1 ) Prepare a test solution containing 10 μM TBCL, and test its absorption spectrum using a UV spectrophotometer. The results are shown in Figure 1 .
[0049] from Figure 1 It can be seen that the organic compound TBCL has a large absorbance value at 350-600nm and two fluorescence peaks at 350-600nm, which shows that the fluorescent compound can be excited by 350-600nm light, and its optimal excitation wavelength is 405nm and 530nm.
[0050] Example 3 Fluorescence test experiment of organic compound TBCL in solutions with different pH values
[0051] DMSO was used to prepare a 1 mM stock solution of the organic compound. The pH of the PBS solution was adjusted with hydrochloric acid and NaOH to prepare solvents with different pH values (pH = 3, 4, 5, 6, 7, 8, 9), and then TBCL was added to prepare a test solution containing 10 μM TBCL.
[0052] The absorption spectrum of the above solution was measured by UV spectrophotometer. Figure 2 The fluorescence emission spectrum was tested with a fluorescence spectrometer, and the corresponding curve was obtained by fitting the fluorescence spectrum. Figure 3 .
[0053] Figure 2 Two absorption peaks appear in solutions with different pH values. As the pH value increases, the absorption peak around 400 nm gradually decreases, while the absorption peak around 530 nm gradually increases, indicating that this organic compound can be used to observe changes in solution pH through UV absorption spectroscopy. The images taken show that the color of the organic small molecule gradually changes from yellow to red as the pH value increases, further demonstrating that organic compounds can be used to observe the pH value of a solution.
[0054] Since the compound has a high degree of structural conjugation, it may produce a significant response to fluorescence. Therefore, we tested the fluorescence spectra of the probe at different pH values. Figure 3 It can be seen that when excited with a single excitation light, two fluorescence peaks appear, namely 530nm and 590nm; as the pH value increases, the fluorescence intensity at 530nm decreases, while the fluorescence intensity at 590nm remains unchanged.
[0055] To further verify that organic compounds can quantify the pH value of the solution, the fluorescence intensity of the organic compounds at 530 and 590 nm was measured. Figure 3 As can be seen in (B), the ratio of the two decreases, and the fitting curve is log[(1.5-I) / (I–0.07)]=pH-5, indicating that the probe can measure the pH value of the solution.
[0056] Example 4: pH of TBCL solution for detecting organic compounds
[0057] The following steps are involved:
[0058] (1) Add TBCL to the test solution so that the concentration of TBCL in the solution is 10 μM;
[0059] (2) using a fluorescence spectrophotometer to excite the solution under a single excitation light of 470 nm to obtain the fluorescence intensity ratio I of the solution at 530 nm and 590 nm;
[0060] (3) The pH value of the mixed solution is directly calculated according to the equation log[(1.5-I) / (I-0.07)]=pH-5.
[0061] The pH value of the solution to be tested was 6.08 when measured with a pH meter, and the pH value calculated using the method of this embodiment was 5.98, indicating that the method of the present invention is reliable and highly sensitive.
[0062] Comparative Example 1 The inventors previously disclosed a compound having the structural formula:
[0063]
[0064] When detecting the pH of a solution, this compound requires two types of excitation light. The specific method is:
[0065] The compound is placed in a cuvette and excited with 405 nm excitation light, and the corresponding spectrum 1 is obtained using a fluorescence spectrometer. The compound is then placed in the instrument and excited with 543 nm light to obtain a fluorescence spectrum 2 under 543 nm excitation light. The two spectrum files are opened separately, and the fluorescence intensity ratio I of the compound in the test solution at 550 nm in spectrum 1 and 605 nm in spectrum 2 is calculated. The pH value is then calculated by substituting the formula (log [(Imax-I) / (I-Imin)] = pH-pKa, where pKa = 5.87, and Imax and Imin are the maximum and minimum values of the ratio in the fitting curve obtained by measuring the known solution.
[0066] By comparison with the method of the present invention, it can be seen that when the method of the present invention is used to measure pH value, the operation is simple, and more importantly, the photodamage of the probe can be reduced due to the reduction in the number of excitations.
[0067] Example 5 Fluorescence test experiment of organic compound TBCL in RNA solutions with different concentrations
[0068] Prepare a 1 mM stock solution of the organic compound using DMSO. Dissolve RNA (derived from calf thymus) at different concentrations (0, 100, 200, 300, 400, 500, 600, 700, 800, 1000, 1200, 1400, 1600, 1800, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000 μM) in tris buffer. Then, add TBCL to prepare a test solution containing 10 μM TBCL. The absorption and fluorescence spectra of the above solutions were measured using a UV-visible spectrophotometer and a fluorescence spectrometer, respectively. For results, see Figure 4 .
[0069] from Figure 4 As can be seen in (A), when RNA is added, the compound absorbs at 420-660 nm, indicating that it can be excited by light in this range. Figure 4 (B) is the fluorescence spectrum of the compound TBCL in solutions with different RNA concentrations. The RNA concentration increases from bottom to top in each curve in the figure. It can be seen from the figure that the fluorescence intensity gradually increases with the increase of RNA concentration. Figure 4 (C) is the fluorescence curve of the probe at 570 nm in solutions with different RNA concentrations. From the fitting curve of Figure 4 (D), it can be seen that its RNA binding constant is 1.08×10 3 M -1 .
[0070] This shows that the organic compound TBCL can be used to detect RNA.
[0071] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes may be made to it in form and detail without departing from the spirit and scope of the present invention as defined in the appended claims.
Claims
1. A benzothiazole organic compound, characterized in that: The chemical name of the compound is (E)-2-(3-chloro-4-hydroxy-5-methoxyphenyl)-3-methylbenzothiazole-3-iodine salt; its structural formula is: 。 2. Use of the benzothiazole organic compound according to claim 1 for non-disease diagnosis and treatment purposes by observing the pH of a solution under single excitation light.
3. The use according to claim 2, characterized in that The compound detects the pH value of the solution under visible light conditions.
4. The use according to claim 2, characterized in that The pH value of the solution is in the range of 3.0-9.
0.
5. The use according to claim 2, characterized in that The concentration of the compound in solution was 5-15 μM.
6. The use according to claim 5, characterized in that The concentration of the compound in solution was 10 μM.
7. A method for detecting pH of a solution of a benzothiazole organic compound for non-disease diagnosis and treatment purposes according to claim 1, characterized in that: The following steps are involved: (1) adding the benzothiazole organic compound to the solution to be tested; (2) using a fluorescence intensity detection device to excite the solution under a single excitation light of 470 nm to obtain a fluorescence intensity ratio I of the solution at 530 nm and 590 nm; (3) Directly calculate the pH value of the test solution according to the equation log[(1.5-I) / (I-0.07)] = pH-5.
8. Use of the benzothiazole organic compound according to claim 1 in the preparation of RNA detection reagents.