A terbium cryptate chelating agent, its preparation method and use

Abstract

The application provides a terbium cryptate chelator, a preparation method and application thereof, and relates to the fields of organic synthesis, rare earth coordination chemistry and immune analysis. The structural formula of the terbium cryptate chelator is shown as formula (I). The chelator is prepared by stirring and refluxing a compound shown as formula (II) with a methanol solution of TbCl3.6H2O in the presence of pyridine to obtain a chelate shown as formula (III), then stirring the chelate shown as formula (III) with a DMF solution of bis-succinimidyl suberate in the presence of triethylamine to obtain the terbium cryptate chelator shown as formula (I). The application introduces active coupling sites and hydrophilic groups into the terbium cryptate complex, so that the terbium cryptate chelator has long fluorescence lifetime, high quantum yield, large molar absorption coefficient, high chelate stability, water solubility and direct bio-covalent labeling capacity. The application also provides application and performance verification of the terbium cryptate chelator in labeling of an antibody Ab630.

Description

Technical Field

[0001] This invention relates to the fields of organic synthesis, rare earth coordination chemistry, and immunoassay, specifically to a terbium cavitation chelating agent, its preparation method, and its application. Background Technology

[0002] Cisbio Bioassays' 50 years of expertise in in vitro immunodiagnostics, combined with its collaboration with Professor Lean-Marie Lehn, a Nobel Prize laureate in Chemistry, on rare earth cavitation structures, has led to the development of homogeneous time-resolved fluorescence immunoassay (TRFIA). Technology. In homogeneous TRFIA technology, terbium cavitating chelators developed by Professor K. Raymond's team at Berkeley have the following significant advantages over europium cavitating chelators due to their unique physicochemical properties and exceptional structural stability: 1. Longer fluorescence lifetime and extremely high signal-to-noise ratio: Terbium cavitary chelating agents have a longer fluorescence lifetime (ms level) than europium cavitary chelating agents (μs level). In biomedical detection that requires ultra-high sensitivity and accurate identification, terbium has become the first choice due to its superior optical performance.

[0003] 2. High energy transfer efficiency: In the “antenna effect” design, terbium-labeled substances have higher efficiency in ligand energy absorption and transfer, making them particularly suitable for building highly sensitive biosensing systems.

[0004] 3. Good spectral compatibility: Terbium-labeled substances, as fluorescent donors, are highly compatible with green dyes, allowing them to be used in dual-labeling systems in conjunction with other green fluorescent molecules (such as fluorescein and GFP) to achieve multi-labeling and multi-parameter detection.

[0005] 4. Suitable for complex biological environments: Terbium-labeled substances have a particularly stable structure and can withstand complex reaction conditions such as DMSO, EDTA, and high ionic strength. Due to their excellent anti-interference ability, they are more commonly used for molecular detection in complex matrices such as serum and cell lysates.

[0006] In the prior art, patent CN114624433A discloses a terbium cavitary chelating agent with amino or carboxyl linkage groups on its side chain. When chelating agents with amino side chains label proteins, the chelating agent can be dissolved in DMF or DMSO and diluted in a buffer solution. Then, the coupling agent dissolved in DMF or DMSO is added and stirred for 1-3 hours. The resulting mixture is then added dropwise to the protein to be labeled. However, due to insufficient activation of the coupling agent, the process is sensitive to pH, unstable, and has a low labeling ratio. On the other hand, chelating agents with carboxyl side chains are dissolved in DMF or DMSO and diluted in a buffer solution. EDC and NHS are then added for the reaction. This reaction lacks active coupling sites and can only perform indirect labeling.

[0007] Patent CN112174988B discloses a trimeric pore ether cage structure, notably featuring a molar extinction coefficient as high as 80,000 M. -1 ·cm -1 This structure is not a small molecule; it suffers from drawbacks such as excessively large molecular weight, significant steric hindrance, and a lack of active coupling sites. Consequently, it cannot be directly labeled and would make labeling more difficult. Summary of the Invention

[0008] The purpose of this invention is to provide a terbium cavitary chelating agent, its preparation method, and its applications. By introducing active coupling sites and hydrophilic groups into terbium cavitary complexes, it achieves a combination of long fluorescence lifetime, high quantum yield, large molar absorptivity, high chelation stability, water solubility, and direct bio-covalent labeling capability. The chelating agent of this invention has a rational synthetic route and high synthesis yield. Its signal-to-noise ratio and sensitivity reach internationally leading levels, filling a gap in the market and providing a domestic alternative to imported Cisbio (Revvity) Lumi4-Tb reagents. This enables applications in multiple fields, including drug screening, disease diagnosis, life science research, immunological analysis (including G protein-coupled receptor research, kinase activity detection, cytokine secretion detection, and antibody detection), freeing researchers from dependence on expensive reagent kits.

[0009] This invention first provides a terbium cavitation chelating agent, with the structural formula shown in formula (I): Formula (I).

[0010] This invention also provides a method for preparing a terbium cavitation chelating agent, comprising: Step 1: The compound shown in formula (II) is reacted with a methanol solution of TbCl3·6H2O in the presence of pyridine under stirring and reflux to obtain the chelate shown in formula (III); Formula (II), Formula (Ⅲ), Step 2: The chelate shown in formula (III) is reacted with a DMF solution of bis(succinimide) octanoate in the presence of triethylamine to obtain the terbium cavitary chelating agent shown in formula (I); Formula (I).

[0011] Preferably, the reaction temperature in step one is 50-65℃, and the reaction time is 6-24h.

[0012] Preferably, the molar ratio of the compound shown in formula (II) in step one to TbCl3·6H2O is 1:1 to 1:5.

[0013] Preferably, the reaction temperature in step two is -10℃ to 10℃, and the reaction time is 0.5-10h.

[0014] Preferably, the molar ratio of the chelate shown in formula (III) in step two to the bis(succinimide) octanoate is 1:1 to 1:10.

[0015] The present invention also provides the use of the above-mentioned terbium cavitary chelating agent in labeling streptavidin or antibody Ab630.

[0016] Preferably, the application method includes: The terbium cavitation chelating agent was dissolved in a buffer solution and stirred to obtain a solution containing the terbium chelating agent. Then, it was added dropwise to a PB buffer solution of streptavidin or antibody Ab630, stirred to react, and separated by dextran gel G25 or G50 column chromatography. The protein characteristic peak at 280 nm was collected to obtain the fluorescent label.

[0017] Preferably, the reaction temperature is 4℃-37℃ and the reaction time is 1-24h.

[0018] Preferably, the molar ratio of the terbium cavitating chelating agent to streptavidin or antibody Ab630 is 20:1-30:1.

[0019] Beneficial effects of the present invention This invention provides a terbium cavitation chelating agent, its preparation method, and its application. The chelating agent has an excitation wavelength of 340 nm, an emission wavelength of 540-560 nm, and a molar extinction coefficient (ε) of 29,830 M. -1 cm -1 With a quantum yield (Φ) of 0.896 and a fluorescence lifetime (τ) of 2.69 ms, this chelating agent exhibits excellent water solubility and biocompatibility. Direct labeling can yield highly bioactive and high-fluorescence-intensity markers. Energy transfer efficiency was compared with that of foreign competitors, and the signal-to-noise ratio and sensitivity reached international leading levels. It can effectively replace imported reagents such as Cisbio Tb-Ab630, freeing users from dependence on expensive reagent kits.

[0020] This invention provides a terbium cavitation chelating agent with a reasonable synthetic route and process, and the product purity is nearly 100% (see...). Figure 2 This has created an upstream (chelating agent) → midstream (label) → downstream (reagent kit) industry chain. This chelating agent is applied not only in drug screening, disease diagnosis, life science research, and immunological analysis, including G protein-coupled receptor research, kinase activity detection, cytokine secretion detection, and antibody detection, but is also particularly suitable for high-throughput drug screening, POCT rapid diagnostics, and CRO platforms. It also has broad market prospects in food safety, environmental monitoring, and drug analysis. Attached Figure Description

[0021] Figure 1This is the mass spectrum of the terbium cavitary chelating agent prepared in Example 1 of the present invention; Figure 2 This is an HPLC chromatogram of the terbium cavitary chelating agent prepared in Example 1 of the present invention; Figure 3 The excitation and emission spectra of the terbium cavitary chelating agent prepared in Example 1 of this invention are shown. Figure 4 The fluorescence lifetime diagram of the terbium cavitary chelating agent prepared in Example 1 of this invention; Figure 5 This is a quantum yield diagram of the terbium cavitation chelating agent prepared in Example 1 of the present invention; Figure 6 This is a comparison chart of energy transfer data between the terbium cavitation chelating agent markers prepared in Examples 3-5 of this invention and the imported markers. Detailed Implementation

[0022] This invention first provides a terbium cavitary chelating agent, named terbium Cryptoate chelating agent, with the structural formula shown in formula (I): Formula (I).

[0023] This invention also provides a method for preparing a terbium cavitation chelating agent, comprising: Step 1: The compound shown in formula (II) is reacted with a methanol solution of TbCl3·6H2O in the presence of pyridine under stirring and reflux. The preferred reaction temperature is 50-65℃, and the reaction time is 6-24h. After cooling, the solvent is removed under vacuum, and the mixture is dried under vacuum. The product is then separated by reverse-phase silica gel column chromatography to obtain the chelate shown in formula (III). The preferred molar ratio of the compound shown in formula (II) to TbCl3·6H2O is 1:1-1:5, and the preferred molar ratio of the compound shown in formula (II) to pyridine is 1:50-1:200. The reaction process is as follows: (II) (III) Step 2: The chelate shown in formula (III) and a DMF solution of bis(succinimide) octanoate are reacted with triethylamine in a stirred reaction. The preferred reaction temperature is -10℃ to 10℃, and the preferred reaction time is 0.5-10 h. Then, diethyl ether is added to separate the solid, which is washed and vacuum dried to obtain terbium cryptotate chelating agent. The preferred molar ratio of the chelate shown in formula (III) to bis(succinimide) octanoate is 1:1-1:10, and the preferred molar ratio of the chelate shown in formula (III) to triethylamine is 1:10-1:50. The reaction process is as follows: (III) (I) The present invention also provides the use of the above-mentioned terbium cavitary chelating agent in labeling streptavidin or antibody Ab630.

[0024] Preferably, the application method includes: The terbium chelating agent is dissolved in a buffer solution, preferably a 0.1M PB pH 7.0-8.5 buffer solution, preferably diluted to 0.5-2 mg / mL. The solution is stirred until homogeneous, preferably at room temperature, and for 0.5-1.0 h to obtain a solution containing the terbium chelating agent. This solution is then added dropwise to a PB buffer solution of streptavidin or antibody Ab630, and the reaction is stirred. The reaction temperature is preferably 4℃-37℃, and the reaction time is preferably 1-24 h. The solution is separated by dextran gel G25 or G50 column chromatography, and the 280 nm protein characteristic peak is collected to obtain a fluorescent label, named terbium cryptate label.

[0025] The present invention will be further described in detail below with reference to specific embodiments.

[0026] Example 1: Preparation of Terbium Cryptorate Chelating Agent Step 1: Dissolve 33.4 mg of the compound shown in formula (II) in 30 mL of methanol, then add 14.9 mg of TbCl3·6H2O, heat to 55 °C under nitrogen protection, add 0.2 mL of pyridine dropwise, continue stirring and reflux for 18 h, cool, and remove the solvent under vacuum. Dry the resulting solid under vacuum, separate by reversed-phase silica gel column chromatography, and obtain the chelate shown in formula (III) with a yield of 93%.

[0027] Step 2: Dissolve 10 mg of the chelate shown in formula (III) in 1.5 mL of DMF, then add 14.4 mg of a DMF solution of bis(succinimide) octanoate (0.5 mL), and add 50 μL of triethylamine dropwise. Stir at -5 °C for 2 h under nitrogen protection, add diethyl ether to precipitate the solid, wash, and vacuum dry to obtain the terbium Cryptotate chelating agent shown in formula (I) with a yield of 75%.

[0028] Figure 1 The mass spectrum of the terbium cryptotate chelating agent prepared in Example 1 of this invention; Figure 2 This is an HPLC chromatogram of the terbium cryptotate chelating agent prepared in Example 1 of this invention; from Figure 2 As can be seen from the data in Table 1, the purity of this chelating agent is 100%.

[0029] Table 1 Processing Channel Description: DAD 340nm Figure 3The excitation spectrum of the terbium cryptotate chelating agent prepared in Example 1 of this invention ( Figure 3 A) and emission spectrum ( Figure 3 B); it can be seen that the chelating agent has an excitation wavelength of 340nm and an emission wavelength of 540-560nm.

[0030] Figure 4 The fluorescence lifetime of the terbium cryptotate chelating agent prepared in Example 1 of this invention is shown in the image (instrument name: research-grade fluorescence spectrometer; model: FLSP 920; manufacturer: EDINBURGH, UK). It can be seen that the fluorescence lifetime of the chelating agent of this invention is 2.69 ms.

[0031] Figure 5 The quantum yield diagram of the terbium cryptotate chelating agent prepared in Example 1 of this invention (instrument name: absolute quantum yield measuring instrument; model: C9920-02; manufacturer: Hamamatsu, Japan); from Figure 5 As can be seen from the data in Table 2, the quantum yield of the chelating agent of the present invention is 0.896.

[0032] Table 2 Tb quantum yield

[0033] Based on the absorbance (Abs) of the terbium cavitation chelate prepared in Example 1, the molar extinction coefficient (ε) was calculated. The absorbance (Abs) of the terbium cavitation chelate measured by this instrument (instrument name: Absolute Quantum Yield Measuring Instrument, Model: C9920-02, Manufacturer: Hamamatsu, Japan) was 0.701. According to Lambert-Beer's law, the molar extinction coefficient (ε) of the terbium cavitation chelate was calculated to be 29,830 M. -1 cm -1 .

[0034] Example 2: Preparation of SA-Tb markers using terbium-containing cryptotate chelating agents 0.1 mg of the terbium-Cryptate chelating agent prepared in Example 1 was dissolved in 0.1 M PB pH 8.5 buffer and diluted to 2 mg / mL. The solution was stirred at room temperature for 0.5 h. 40 μL of the solution containing the terbium-Cryptate chelating agent was added dropwise to 0.1 mg of SA in PB buffer solution, and the reaction was stirred at 4 °C for 24 h. Separation was performed using a dextran gel G25 column chromatography, and the characteristic protein peak at 280 nm was collected to obtain the SA-Tb fluorescent label.

[0035] Example 3: Preparation of Ab630-Tb markers using terbium cryptotate chelating agents 0.1 mg of the terbium cryptotate chelating agent prepared in Example 1 was dissolved in 0.1 M PB pH 8.0 buffer and diluted to 1 mg / mL. The solution was stirred at room temperature for 1.0 h. 50 μL of the solution containing the terbium cryptotate chelating agent was added dropwise to 0.2 mg of Ab630 in PB buffer solution and stirred at 37 °C for 1 h. The protein was separated using a dextran gel G25 column, and the characteristic peak at 280 nm was collected to obtain the Ab630-Tb fluorescent label, denoted as Tb Ab-1.

[0036] Example 4: Preparation of Ab630-Tb markers using terbium cryptotate chelating agents 0.1 mg of the terbium cryptotate chelating agent prepared in Example 1 was dissolved in 0.1 M PB pH 7.5 buffer and diluted to 1.5 mg / mL. The solution was stirred at room temperature for 0.5 h. 30 μL of the solution containing the terbium cryptotate chelating agent was added dropwise to 0.2 mg of Ab630 in PB buffer solution and stirred at room temperature for 3 h. The protein was separated using a dextran gel G25 column, and the characteristic peak at 280 nm was collected to obtain the Ab630-Tb fluorescent label, denoted as Tb Ab-2.

[0037] Example 5: Preparation of Ab630-Tb markers using terbium cryptotate chelating agents 0.1 mg of the terbium cryptotate chelating agent prepared in Example 1 was dissolved in 0.1 M PB pH 7.2 buffer and diluted to 0.5 mg / mL. The solution was stirred at room temperature for 0.5 h. 120 μL of the solution containing the terbium cryptotate chelating agent was added dropwise to 0.2 mg of Ab630 in PB buffer solution and stirred at room temperature for 4 h. The protein was separated using a dextran gel G25 column, and the characteristic peak at 280 nm was collected to obtain the Ab630-Tb fluorescent label, denoted as Tb Ab-3.

[0038] Energy transfer experimental data were compared between the terbium cryptotate marker of this invention and the imported marker (K15, Chinese name Cisbio Tb-Ab630 marker), and the results are as follows: Figure 6 As shown in Figure 1 and Table 3, it can be seen from the figure that the energy transfer efficiency of the Ab630-Tb fluorescent marker prepared in this invention for high-throughput drug screening surpasses that of international benchmark products.

[0039] Table 3 Comparison of Terbium Cryptorate Marker Data with Imported Markers The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

[0040] Furthermore, those skilled in the art will understand that although some embodiments herein include certain features included in other embodiments but not others, combinations of features from different embodiments are meant to be within the scope of the invention and form different embodiments. For example, in the foregoing claims, any of the claimed embodiments can be used in any combination. The information disclosed in this background section is intended only to enhance the understanding of the general background of the invention and should not be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

Claims

1. A terbium cavitation chelating agent, characterized in that, The structural formula is shown in equation (I): Equation (I).

2. The method for preparing a terbium cavitation chelating agent according to claim 1, characterized in that, include: Step 1: The compound shown in formula (II) is reacted with a methanol solution of TbCl3·6H2O in the presence of pyridine under stirring and reflux to obtain the chelate shown in formula (III); Formula (II), Formula (Ⅲ), Step 2: The chelate shown in formula (III) is reacted with a DMF solution of bis(succinimide) octanoate in the presence of triethylamine to obtain the terbium cavitary chelating agent shown in formula (I); Equation (I).

3. The method for preparing a terbium cavitation chelating agent according to claim 2, characterized in that, The reaction temperature in step one is 50-65℃, and the reaction time is 6-24h.

4. The method for preparing a terbium cavitation chelating agent according to claim 2, characterized in that, The molar ratio of the compound shown in formula (II) in step one to TbCl3·6H2O is 1:1-1:

5.

5. The method for preparing a terbium cavitation chelating agent according to claim 2, characterized in that, The reaction temperature in step two is -10℃ to 10℃, and the reaction time is 0.5-10h.

6. The method for preparing a terbium cavitation chelating agent according to claim 2, characterized in that, The molar ratio of the chelate shown in formula (III) in step two to the bis(succinimide) octanoate is 1:1 to 1:

10.

7. The use of the terbium cavitary chelating agent according to claim 1 in labeling streptavidin or antibody Ab630.

8. The application according to claim 7, characterized in that, The application method includes: The terbium cavitation chelating agent was dissolved in a buffer solution and stirred to obtain a solution containing the terbium chelating agent. Then, it was added dropwise to a PB buffer solution of streptavidin or antibody Ab630, stirred to react, and separated by dextran gel G25 or G50 column chromatography. The protein characteristic peak at 280 nm was collected to obtain the fluorescent label.

9. The application according to claim 8, characterized in that, The reaction temperature is 4℃-37℃, and the reaction time is 1-24h.

10. The application according to claim 8, characterized in that, The molar ratio of the terbium cavitating chelating agent to streptavidin or antibody Ab630 is 20:1-30:1.

Images