A terbium complex, a fluorescent dye and a synthesis method and application thereof

Abstract

The application discloses a terbium complex, a fluorescent dye and a synthesis method and application thereof. The terbium complex has a structure shown in formula (I), wherein n is an integer from 9 to 227. When the terbium complex is used as a lipoprotein dyeing agent, specific marking of lipoprotein and sub-components thereof can be realized through long-life fluorescent emission of the rare earth terbium complex and specific interaction with lipoprotein components, the terbium complex has the advantages of large stokes shift and being applicable to time-resolved imaging, is suitable for trace biological sample detection, can realize detection of low-density lipoprotein sub-components through trace fingertip blood sample detection, and provides a new fluorescent dye for specific detection of lipoprotein and sub-components thereof.

Description

Technical Field

[0001] This application relates to the field of fluorescent dye technology, and in particular to a terbium complex, a fluorescent dye, a method for synthesizing the same, and its applications. Background Technology

[0002] Cardiovascular disease is one of the leading causes of death worldwide, making early and accurate risk assessment crucial. Low-density lipoprotein (LDL) cholesterol is widely recognized as a key biomarker for atherosclerotic cardiovascular disease. Notably, LDL exhibits high heterogeneity in particle size, density, chemical composition, and its atherogenic properties. Therefore, enabling convenient detection of different LDL subfractions is of significant practical importance for promoting early disease screening and personalized treatment.

[0003] Currently, the analysis of lipoprotein subfractions mainly relies on several high-precision physicochemical methods: (1) Nuclear magnetic resonance spectroscopy, which uses the lipid methyl signal characteristics of lipoprotein particles for quantification, has the advantages of being non-destructive and having high throughput, and is one of the mainstream technologies in clinical research, but the equipment is expensive and costly. (2) Density gradient ultracentrifugation and high performance liquid chromatography are the "gold standard" methods for separating and quantifying lipoprotein subfractions of different densities, with high separation accuracy, but the operation is cumbersome, time-consuming, and has low throughput, making it difficult to meet the needs of large-scale clinical screening. (3) Ion mobility analysis and mass spectrometry can provide multi-dimensional information such as particle size and charge, and is a cutting-edge direction for accurate typing, but it also faces the problems of high instrument cost and complex operation. Although the above-mentioned existing technologies have high accuracy, they generally suffer from bottlenecks such as expensive equipment, complex operation, long detection cycle, high professional requirements for operators, and difficulty in promotion in primary medical institutions, which limit their application in widespread clinical screening.

[0004] To overcome these shortcomings, there is an urgent need to develop a novel detection technology that is easy to operate while maintaining high sensitivity. Fluorescence detection, due to its high sensitivity, relatively simple equipment, and ease of rapid detection and automation, is an ideal candidate technology. However, conventional organic fluorescent dyes often face problems such as large background fluorescence interference, poor photostability, and insufficient lipid-specific recognition ability when used on complex biological samples such as whole blood.

[0005] Rare-earth terbium complex fluorescent dyes possess unique photophysical properties, such as large Stokes shift, sharp emission peaks, and long fluorescence lifetimes. These properties allow for effective filtering of short-lived background fluorescence interference using time-resolved fluorescence detection techniques, making them particularly suitable for analyzing complex biological samples. However, there are currently no studies or reports on the development of terbium complexes as fluorescent dyes for the specific detection of lipoprotein subfractions. Summary of the Invention

[0006] The purpose of this application is to provide a novel terbium complex, a fluorescent dye based on the terbium complex, and a method for synthesizing the terbium complex and the fluorescent dye, as well as their applications.

[0007] To achieve the above objectives, this application adopts the following technical solution:

[0008] A first aspect of this application discloses a terbium complex having the structure shown in formula (I).

[0009] ,

[0010] Where n takes the value of an integer from 9 to 227. Where the value of n is such that the number average molecular weight of polyethylene glycol is in the range of 400 to 10000, for example PEG2000.

[0011] It should be noted that the terbium complex with the structure shown in formula (I) of this application, when used as a lipoprotein staining agent, achieves highly sensitive labeling of lipoprotein subcomponents through the long-lifetime fluorescence emission of the rare earth terbium complex and its specific interaction with lipoprotein components. The terbium complex of this application has the advantages of large Stokes shift, low background interference, high signal-to-noise ratio, and good repeatability, and is suitable for the detection of trace biological samples. For example, by applying the terbium complex of this application to finger-prick blood samples, the detection of low-density lipoprotein (LDL) and its subcomponents can be achieved under the condition that the blood collection volume does not exceed 50 μL, such as in some experiments where only 2 μL of serum sample is required.

[0012] A second aspect of this application discloses a fluorescent dye, which includes the terbium complex of this application.

[0013] In one implementation of this application, the fluorescent dye is formed by dissolving the terbium complex of this application in an organic solvent.

[0014] In one implementation of this application, the organic solvent for the fluorescent dye is propylene glycol.

[0015] It should be noted that the key to the fluorescent dye in the embodiments of this application lies in the use of the terbium complex of this application; therefore, it can specifically and sensitively label lipoprotein subfractions, and also has the advantages of large Stokes shift, low background interference, high signal-to-noise ratio, good repeatability, and suitability for detection of trace biological samples.

[0016] The third aspect of this application discloses the use of terbium complexes or fluorescent dyes from the embodiments of this application in the preparation of detection reagents.

[0017] In one implementation of this application, the detection reagent includes a reagent for detecting lipoproteins.

[0018] A fourth aspect of this application discloses a detection reagent or detection kit containing the terbium complex or the fluorescent dye of this application.

[0019] It should be noted that the terbium complex or fluorescent dye of this application embodiment can specifically and sensitively label lipoprotein subfractions; therefore, they can be used to prepare lipoprotein detection reagents or kits. Lipoproteins particularly include low-density lipoprotein and its subfractions.

[0020] In one implementation of this application, the detection reagent or detection kit includes a method for detecting lipoproteins.

[0021] The fifth aspect of this application discloses a method for preparing the terbium complex of this application, comprising the following steps:

[0022] (a) Provide intermediate compound 4;

[0023] (b) The intermediate compound 4 from step (a) is reacted with a trivalent terbium compound to obtain the terbium complex of this application, namely the terbium complex with the structure shown in formula (I);

[0024] Compound 4

[0025] ,

[0026] In compound 4, n ranges from 9 to 227. The value of n ensures that the number-average molecular weight of the polyethylene glycol in compound 4 ranges from 400 to 10000, for example, PEG2000.

[0027] In one implementation of this application, step (a), the method for preparing intermediate compound 4 includes: reacting compound 2 and compound 3 through an amide condensation reaction to obtain intermediate compound 4;

[0028] Compound 2 ,

[0029] Compound 3 ,

[0030] In compound 3, n takes the value of an integer from 9 to 227.

[0031] In one implementation of this application, step (a) the amide condensation reaction is carried out in an organic solvent.

[0032] In one implementation of this application, the organic solvent used in the condensation reaction is selected from at least one of dimethylformamide, dichloromethane, methanol, and ethanol.

[0033] In one implementation of this application, step (a) amide condensation reaction is carried out at 30~100 °C.

[0034] In one implementation of this application, step (a) the amide condensation reaction is carried out under alkaline conditions.

[0035] In one implementation of this application, the alkali is selected from at least one of sodium hydroxide, potassium hydroxide, potassium carbonate, triethylamine, and N,N-diisopropylethylamine (DIPEA).

[0036] In one implementation of this application, the trivalent terbium compound in step (b) includes at least one of terbium(III) acetate hydrate, terbium chloride hydrate, and terbium nitrate.

[0037] In one implementation of this application, the reaction in step (b) is carried out under the catalysis of piperidine and / or pyridine.

[0038] In one implementation of this application, the reaction in step (b) is carried out in a solvent.

[0039] In one implementation of this application, the solvent in step (b) includes at least one of methanol and dichloromethane.

[0040] In one implementation of this application, the reaction in step (b) is carried out at 20–60 °C. For example, step (b) uses pyridine as a catalyst and is carried out in methanol solvent at room temperature.

[0041] A sixth aspect of this application discloses an intermediate having the structure shown in formula (II).

[0042] ,

[0043] Wherein, the value of n ranges from 9 to 227. Wherein, the value of n is such that the number average molecular weight of polyethylene glycol in the structure shown in formula (II) is in the range of 400 to 10000, for example, PEG2000.

[0044] It should be noted that the intermediate with the structure shown in formula (II) of the present application embodiment is actually compound 4 in the preparation method of the terbium complex of the present application embodiment. This intermediate can be used as an independent product to prepare the terbium complex of the present application through step (b) of the preparation method of the terbium complex of the present application. As for the preparation method of the intermediate of the present application, it can be referred to step (a) of the preparation method of the terbium complex of the present application, and will not be repeated here.

[0045] The seventh aspect of this application discloses an analytical method for low-density lipoprotein and its subcomponents, which includes staining the sample to be tested with the terbium complex, the fluorescent dye, or the kit of this application, and analyzing the low-density lipoprotein or its subcomponents in the sample to be tested based on the staining results.

[0046] It should be noted that the analytical method for low-density lipoprotein and its subcomponents in this application uses in vitro samples as the detection object. Furthermore, the direct detection results obtained are only qualitative and / or quantitative results of low-density lipoprotein or its subcomponents, and do not directly reveal the disease status of the organism providing the test sample. Therefore, the analytical method for low-density lipoprotein and its subcomponents in this application is not a disease diagnosis method.

[0047] Due to the adoption of the above technical solutions, the beneficial effects of the embodiments of this application are as follows:

[0048] When the terbium complexes of this application are used as lipoprotein staining agents, they can achieve specific labeling of lipoproteins and their subcomponents through the long-lifetime fluorescence emission of rare earth terbium complexes and their specific interaction with lipoprotein components. They have the advantages of large Stokes shift and can be used for time-resolved imaging. They are also suitable for the detection of trace biological samples, and can detect low-density lipoprotein subcomponents through the detection of trace finger-prick blood samples, providing a new fluorescent dye for the specific detection of lipoproteins or their subcomponents. Attached Figure Description

[0049] Figure 1 The absorption spectrum of the terbium complex of formula (I) in the embodiments of this application is shown below;

[0050] Figure 2 The emission spectrum of the terbium complex of formula (I) in the embodiments of this application;

[0051] Figure 3 This is an image showing the results of separating serum lipoproteins using the gel electrophoresis system in this embodiment of the application. Detailed Implementation

[0052] Rare-earth terbium complex fluorescent dyes possess unique photophysical properties, such as large Stokes shift, sharp emission peaks, and long fluorescence lifetimes. These properties allow for effective filtering of short-lived background fluorescence interference using time-resolved fluorescence detection techniques, making them particularly suitable for analyzing complex biological samples. However, there are currently no studies or reports on the development of terbium complexes as fluorescent dyes for the specific detection of lipoprotein subfractions.

[0053] If a terbium complex fluorescent probe that can target lipoproteins, especially LDL and its atherosclerotic subtypes, can be designed and synthesized, it will be possible to construct a new, convenient, point-of-care or rapid clinical detection method for lipoprotein subfractions based on small amounts of blood samples, which will have important clinical application value for promoting the early prevention of cardiovascular diseases.

[0054] Based on the above research and understanding, this application has creatively developed a new terbium complex, namely the terbium complex with the structure shown in formula (I).

[0055] The terbium complexes of this application possess excellent lipid binding capacity and fluorescence signal characteristics, making them particularly suitable for the identification and imaging of lipid structures in complex biological samples. In detection, the terbium complexes of this application act as highly specific fluorescent dyes, achieving sensitive labeling and quantitative analysis of target components through selective interactions with lipoprotein subfractions.

[0056] The terbium complexes provided in this application and their application as fluorescent probes enable the construction of a convenient and low-cost detection method, requiring extremely low sample volumes, with blood collection volume reduced to approximately 50 microliters, significantly alleviating the sampling burden on subjects. The terbium complexes in this application are expected to substantially improve the level of routine blood lipid monitoring and early risk assessment in large-scale populations, providing key technical support for early warning and proactive intervention of cardiovascular events, and playing a significant role in reducing the incidence and mortality of related diseases.

[0057] The present application will now be described in further detail through specific embodiments. In these embodiments, many details are described to facilitate a better understanding of the present application. However, those skilled in the art will readily recognize that some features may be omitted in different situations, or may be replaced by other devices, materials, or methods. In some cases, certain operations related to the present application are not shown or described in the specification to avoid obscuring the core parts of the application with excessive description. For those skilled in the art, detailed description of these related operations is not necessary; a complete understanding of the related operations can be obtained from the descriptions in the specification and general technical knowledge in the art.

[0058] Unless otherwise specified, all reagents used in the following examples and control examples were purchased from Sigma-Aldrich, with a purity of ≥99%, and were used directly in the experiments without further purification.

[0059] Example 1

[0060] The technical route for preparing intermediate compound 4 is as follows:

[0061]

[0062] Compound 2 (66.0 mg, 50 μmol) was dissolved in 5 mL of anhydrous N,N-dimethylformamide (DMF), followed by the sequential addition of N,N-diisopropylethylamine (DIPEA, 32.3 mg, 250 μmol) and compound 3 (148 mg, 55 μmol) (Yusi Pharmaceutical, catalog number YUSI-D1524005-5mg-PEG2000). The reaction mixture was stirred at 50 °C, and the reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was complete, the reaction solution was cooled to room temperature, an appropriate amount of water was added, and extraction was performed with dichloromethane (3 × 10 mL). The organic phases were combined, washed sequentially with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. Finally, the crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate gradient system) to obtain 143 mg of yellow solid product, with a yield of 71%, which is compound 4. Compound 2 was synthesized according to the method disclosed in Nat. Chem. Biol. 2021, 17, 1168-1177.

[0063] Example 2

[0064] The preparation of the terbium complex with the structure shown in formula (I) follows the following technical route:

[0065]

[0066] Intermediate compound 4 (50 mg, 12 μmol) was dissolved in 200 mL of methanol, and 50 mg of pyridine was added. The mixture was stirred at room temperature for 5 minutes. Separately, 9.4 mg of terbium trichloride hexahydrate (Jieshikai, catalog number KA632192-5g) was dissolved in 10 mL of methanol to prepare a clear solution. Under stirring, this solution was slowly added dropwise to the above reaction system. After the addition was complete, the mixture was stirred at room temperature for another 30 minutes. After the reaction was completed, the methanol solvent was removed under reduced pressure in a rotary evaporator to obtain the crude product. The crude product was purified by column chromatography to obtain 43 mg of a yellow solid product, with a yield of 78%, which is the terbium complex with the structure shown in formula (I).

[0067] The synthesized terbium complex was configured as 10 -6 The concentration of M was analyzed using a real-time PCR instrument in 0.1 M Tris buffer (10% DMSO, pH = 7.4) to determine its absorption spectrum and excitation wavelength. The results are as follows: Figure 1 and Figure 2 As shown. Figure 1 The absorption spectrum of the terbium complex is shown. Figure 2 The emission spectrum of the terbium complex is shown.

[0068] Example 3

[0069] The terbium complex of formula (I) prepared in Example 2 was used for the specific detection of lipoprotein subfractions in finger-prick blood, specifically including:

[0070] This study used polyacrylamide tube gel electrophoresis (PTGE) to separate lipoprotein subclasses. First, terbium complexes were dissolved in propylene glycol to prepare a staining working solution with a concentration of 0.1 mg / mL. Staining procedure: 10 μL of serum sample was taken, and 1 μL of the above staining solution was added. After vortexing and mixing, the mixture was incubated in a 37 ℃ water bath for 5 minutes to complete the lipoprotein labeling. The labeled mixture was centrifuged at 3000 r / min for 3 minutes, and the supernatant was collected to obtain the stained serum.

[0071] The serum used was obtained from venous blood samples collected from volunteers after a 12-hour fast. Serum was separated within 2 hours of blood collection. After removing the surface and preservation solution from the PTGE gel column, it was vertically fixed in a disc electrophoresis tank, with the first electrophoretic separation section on the negative electrode side and the second electrophoretic separation section on the positive electrode side. 500 mL of electrophoresis buffer was added to each tank, and 2.2 μL of stained serum was smoothly added to the surface of the first separation section gel. Electrophoresis was performed at a constant current of 3 mA / tube (3 mA for a single tube system) for 90 minutes. After electrophoresis, the gel column was removed, and images were acquired using an imaging system in time-resolved fluorescence mode. The images were then imported into analysis software for processing, enabling the detection of lipoprotein subfractions in finger-prick blood.

[0072] The image showing the results of separating serum lipoproteins using the gel electrophoresis system in this example is shown below. Figure 3 As shown. Figure 3 The results showed that low-density lipoproteins LDL 1 to LDL 4 were all distributed in the second electrophoretic separation section of the gel column, indicating that the system can effectively separate the major LDL subtypes.

[0073] The above description, in conjunction with specific embodiments, provides a further detailed explanation of this application and should not be construed as limiting the specific implementation of this application to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of this application.

Claims

1. A terbium complex, characterized by: The terbium complex has the structure shown in formula (I). ， Where n takes the value of an integer from 9 to 227.

2. The terbium complex according to claim 1, characterized in that: The value of n is chosen to make the number average molecular weight of polyethylene glycol range from 400 to 10000.

3. A fluorescent dye characterized in that: The fluorescent dye includes the terbium complex as described in claim 1 or 2.

4. The fluorescent dye according to claim 3, characterized in that: The fluorescent dye is formed by dissolving the terbium complex of claim 1 or 2 in an organic solvent; Optionally, the organic solvent includes propylene glycol.

5. The use of the terbium complex according to claim 1 or 2 or the fluorescent dye according to claim 3 or 4 in the preparation of detection reagents; Optionally, the detection reagent is used to detect lipoproteins.

6. A detection reagent or detection kit, characterized in that: Contains the terbium complex as described in claim 1 or 2 or the fluorescent dye as described in claim 3 or 4; Optionally, the detection reagent or kit is used to detect lipoproteins.

7. The method for preparing the terbium complex according to claim 1 or 2, characterized in that: Includes the following steps, (a) Provide intermediate compound 4; (b) The intermediate compound 4 from step (a) is reacted with a trivalent terbium compound to obtain the terbium complex; Compound 4 ， In the intermediate compound 4, n takes the value of an integer from 9 to 227.

8. The preparation method according to claim 7, characterized in that: In step (a), the preparation method of the intermediate compound 4 includes: reacting compound 2 and compound 3 by an amide condensation reaction to obtain the intermediate compound 4; Compound 2 , Compound 3 , In compound 3, n takes the value of an integer from 9 to 227; Optionally, the amide condensation reaction in step (a) is carried out in an organic solvent; Optionally, the amide condensation reaction in step (a) is carried out at 30~100 °C; Optionally, the amide condensation reaction in step (a) is carried out under alkaline conditions; Optionally, the organic solvent used in the condensation reaction includes at least one of dimethylformamide, dichloromethane, methanol, and ethanol; Optionally, the alkali includes at least one selected from sodium hydroxide, potassium hydroxide, potassium carbonate, triethylamine, and N,N-diisopropylethylamine; Optionally, the trivalent terbium compound in step (b) includes at least one of terbium(III) acetate hydrate, terbium chloride hydrate, and terbium nitrate; And / or, the reaction in step (b) is carried out under the catalysis of piperidine and / or pyridine; And / or, the reaction in step (b) is carried out in a solvent; And / or, in the reaction of step (b), the solvent includes at least one of methanol and dichloromethane; And / or, the reaction in step (b) is carried out at 20~60 °C.

9. An intermediate having the structure shown in formula (II), ， in, The value of n ranges from 9 to 227; Optionally, the intermediate is used to prepare the terbium complex according to claim 1 or 2.

10. A method for analyzing a lipoprotein or its subcomponents, characterized in that: This includes staining the test sample with the terbium complex of claim 1 or 2, the fluorescent dye of claim 3 or 4, or the detection reagent or detection kit of claim 6, and analyzing the lipoproteins or their sub-components in the test sample based on the staining results; Optionally, the lipoprotein includes low-density lipoprotein.

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