A near-infrared second region organic small molecule chemiluminescent probe and its preparation and application

Abstract

The present invention provides a near-infrared zone II organic small molecule chemiluminescent probe and its preparation and application, and relates to the technical field of chemiluminescent probe synthesis. The organic small molecule chemiluminescent probe is CD-950, and the luminescent probe is a chemiluminescent probe with a m-dinitrophenol structure as the H2S response site; a Schaap's dioxetane emitting 700nm as the chemiluminescent donor molecule; a fluorophore with a D-A-D skeleton structure with a fluorescence emission at 970nm as the energy acceptor, and the two are bonded together through an amide bond to obtain. The probe obtained by the present invention uses a bio-responsive near-infrared zone II chemiluminescent signal (950nm) for the first time to achieve specific response and detection of H2S in vitro and in cells, and at the same time achieves specific response imaging of H2S highly expressed in metformin-induced mouse liver damage disease; the probe can also achieve in vivo responsive near-infrared zone II chemiluminescent imaging of H2S molecules associated with other diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of chemiluminescent probe synthesis, and in particular to a near-infrared second-region organic small molecule chemiluminescent probe and preparation and application thereof. Background Art

[0002] Chemiluminescence imaging is a luminescence imaging method that converts chemical energy into light energy. Chemiluminescence imaging technology has the advantages of not requiring real-time excitation light, avoiding biological autofluorescence, low imaging background, high signal-to-noise ratio, good tissue penetration and high sensitivity. It provides an effective technical analysis method for obtaining molecular information at the cell and living body level with high sensitivity, specificity and real-time. It has been widely used in research on cell tracking, biosensing, cancer diagnosis and treatment. However, the wavelength range emitted by current chemiluminescent probes is mostly concentrated in the visible light region (Visible range, 400-700nm) and the first near-infrared region (NIR-I, 700-900nm). Because the photons in the above bands are easily absorbed by cells (melanin, hemoglobin, etc.) or living tissues, the current chemiluminescent probes have problems such as low signal-to-noise ratio and poor tissue penetration depth. In recent years, scientists have discovered that photons with wavelengths in the second near-infrared region (NIR-II, 900-1700nm) are less scattered by biological tissues (muscles, bones, skin, mucus, etc.) and less absorbed by biological macromolecules (hemoglobin, blood sugar, pigments in the skin, myoglobin, cytochrome, etc.). In addition, in the NIR-II imaging window, the autofluorescence from various pigments in the body is also greatly reduced. With the help of these two advantages, NIR-II chemiluminescence imaging at the in vivo level can achieve high-resolution, high signal-to-noise ratio, and deep tissue penetration imaging effects.

[0003] However, there are only two reported near-infrared zone II chemiluminescent probes (Angew.Chem.Int.Ed.Engl.59,18380-18385(2020);Chem.Commun.48,6426-6428(2012)), which mainly use hydrophilic polymers to simply physically encapsulate the two components (chemiluminescent donor and fluorophore acceptor) to form nanoparticles, and then use the chemiluminescence resonance energy transfer strategy (Chemiluminescence Resonance Energy Transfer, CRET) between the donor and the acceptor to achieve the red shift of the luminescence wavelength to reach the near-infrared zone II. Because it involves energy conversion between multiple components, after the formation of nanoparticles, the distance between the donor and the acceptor cannot be effectively controlled, the energy conversion efficiency is low, and the non-radiative loss is obvious, which seriously reduces the resolution and penetration depth of imaging.

[0004] Currently, single-molecule-based near-infrared region II chemiluminescent probes for in vivo imaging have not been reported. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a near-infrared second region organic small molecule chemiluminescent probe and its preparation and application, which can effectively detect H 2 S achieves specific response and detection.

[0006] To achieve the above objectives, the technical solution of the present invention is implemented through the following technical solutions:

[0007] A near-infrared region II organic small molecule chemiluminescent probe, the organic small molecule chemiluminescent probe is CD-950, the maximum emission wavelength of which is 950nm, and the structural formula of CD-950 is as follows:

[0008]

[0009] Preferably, the organic small molecule chemiluminescent probe is a probe with m-dinitrophenol structure as H 2 S response site; Schaap's dioxetane emitting at 700nm is used as the donor molecule of chemiluminescence; a fluorophore with a DAD skeleton structure emitting at 970nm is used as the energy acceptor, and the two are linked together through an amide bond to obtain.

[0010] The preparation method of the near-infrared second region organic small molecule chemiluminescent probe comprises the following steps:

[0011] (1) Using 2-bromo-5-hydroxybenzaldehyde as the starting material, a H 2 S-responsive chemiluminescent probe C-700;

[0012] (2) Using 4-bromotriphenylamine as the starting material, the dye molecule D-970 with fluorescence emission at 970 nm was synthesized;

[0013] (3) Synthesis of near-infrared zone II chemiluminescent molecules: The above C-700 and D-970 were subjected to an amidation synthesis method to obtain an amidation product in one step. The compound was then exposed to light at 595 nm for 3 hours in an oxygen atmosphere and in the presence of a catalytic amount of methylene blue to obtain the final target product, an organic small molecule chemiluminescent probe CD-950.

[0014] Preferably, the synthesis steps of C-700 in step (1) are as follows:

[0015]

[0016] Preferably, the synthesis steps in step (2) are as follows:

[0017]

[0018] Preferably, the synthesis steps in step (3) are as follows:

[0019]

[0020] The application of organic small molecule chemiluminescent probes in the near-infrared region II is to use this probe to detect H 2 The specific response of S.

[0021] Preferably, the application is to use the probe to detect H1N1 overexpressed in a metformin-induced liver injury mouse model. 2 Specific detection of S.

[0022] Preferably, the application is to use the probe to detect various disease-related characteristic molecules H in vivo. 2 Specific imaging and detection of S.

[0023] The present invention provides a near-infrared second-region organic small molecule chemiluminescent probe and its preparation and application, which has the following advantages compared with the prior art:

[0024] (1) This probe uses the bioresponsive near-infrared second-region chemiluminescence signal to detect H in vitro and in cells for the first time. 2 S achieved specific response and detection, and also detected H1N1, which was highly expressed in metformin-induced liver injury in mice. 2 This probe can also be used to image H associated with other diseases. 2 In vivo responsive near-infrared second-zone chemiluminescence imaging of S molecules.

[0025] (2) The present invention does not require a real-time excitation light source and can directly detect H in vivo and in vitro using near-infrared second-zone chemiluminescence signals. 2 The concentration change of S can achieve the convenience and accuracy of detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The chemiluminescent molecular probe CD-950 of Example 1 of the present invention 1 HNMR data graph;

[0027] Figure 2 The chemiluminescent molecular probe CD-950 of Example 1 of the present invention 13 CNMR data map;

[0028] Figure 3 CD-950 in Example 2 of the present invention is 2Near-infrared second-zone chemiluminescence spectrum after S addition;

[0029] Figure 4 The near-infrared second-region chemiluminescence and near-infrared second-region fluorescence imaging signals of CD-950 after cell incubation in Example 2 of the present invention;

[0030] Figure 5 The near-infrared second-zone chemiluminescent imaging signal and its intensity change generated inside the tumor of tumor-bearing mice injected with CD-950 in situ in Example 2 of the present invention;

[0031] Figure 6 It is the near-infrared second-zone chemiluminescent imaging signal generated in mice with liver damage after tail vein injection of CD-950 in Example 2 of the present invention. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention is clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] Embodiment 1:

[0034] The synthesis scheme of the near-infrared second-region chemiluminescent probe CD-950 is as follows:

[0035] (1) Based on Schaap's dioxetane structure 2 Synthesis of S-responsive chemiluminescent probe (C-700): A multi-step chemical synthesis method was used, with 2-bromo-5-hydroxybenzaldehyde as the starting material. After 12 steps of chemical reactions, the chemical C-700 was finally obtained (the 12-step synthesis scheme is shown below), which was finally a yellow solid sample.

[0036]

[0037] (2) Synthesis of dye molecule D-970: Using 4-bromotriphenylamine as the starting material, the synthesis of the dye molecule D-970 with fluorescence emission at 970 nm was achieved through a four-step chemical reaction. The specific four-step chemical reaction is as follows:

[0038]

[0039] (3) Synthesis of near-infrared second-region chemiluminescent molecules: First, the amidation product is obtained by one-step chemical synthesis using the classic amidation synthesis method. Then, the compound is illuminated for 3 hours using a 595 nm light source in an oxygen atmosphere and in the presence of a catalytic amount of methylene blue to obtain the final target product CD-950. The specific synthesis steps are as follows:

[0040]

[0041] Detection of the compound CD-950 prepared above 1 HNMR and 13 CNMR data such as Figure 1 , Figure 2 shown.

[0042] Embodiment 2:

[0043] Application testing of the above CD-950:

[0044] 1. Preparation before testing:

[0045] (1) The probe CD-950 was dissolved in a mixture of DMSO and PBS to prepare a stock solution of a certain concentration, which was stored in a -20°C refrigerator away from light and used as needed.

[0046] (2) For in vitro imaging experiments, only the H 2 The standard solution of S can be added into the probe system diluted to a certain concentration to detect the near-infrared second zone chemiluminescence signal in real time.

[0047] (3) For cell experiments, the cells need to be incubated in culture medium containing a certain concentration of probes before imaging to observe the intracellular chemiluminescent signals.

[0048] (4) For intratumor imaging experiments, the probe only needs to be injected in situ into the tumor of a tumor-bearing mouse transplanted with special cancer cells, and then the chemiluminescent signal can be observed in real time.

[0049] (5) For the metformin-induced liver injury experiment in mice, the mouse model was first established successfully according to the literature method, and then the probe was injected through the tail vein. The mice were immediately placed in a near-infrared zone II small animal imager for real-time in vivo imaging analysis.

[0050] 2. In vitro H 2 Specific response and detection of S:

[0051] Take a certain amount of DMSO (10%) / PBS solution containing the probe (50 μM) prepared in advance, and then add the H 2 S (20 μM) standard solution (with a certain concentration of Na 2S was replaced), the above system was added, and then real-time imaging was performed.

[0052] The test results are attached. Figure 3 The chemiluminescence spectrum data showed that after adding H 2 After S, the second zone detector collected the chemiluminescence spectrum of the probe CD-950, whose maximum emission peak was at 950nm, and its intensity was approximately 2 32 times of S.

[0053] 3. Intracellular H 2 Specific response and detection of S:

[0054] First, the probe (50 μM) was prepared into a certain concentration of cell culture medium, and then MC38-Luc, MCF-7, LO 2 The three types of cells were cultured in the culture medium. After 30 minutes, the culture medium on the cell surface was washed off with PBS, and the cells were transferred to a 96-well plate for imaging analysis in a two-zone imager. The imaging results showed that among the three types of cells, only MC38-Luc had obvious chemiluminescent signals, while the other two types of cells had almost no obvious luminescent signals, further indicating that CD-950 can specifically respond to H-expressed proteins in tumor cells. 2 S (see attached Figure 4 ). The near-infrared second zone chemiluminescence signal acquisition uses a 1000nm long-pass lens with an exposure time of 900ms. The near-infrared second zone fluorescence signal acquisition uses a 1000nm long-pass lens, and the laser excitation light source is an 808nm laser (2W / cm 2 ), the exposure time is 50ms.

[0055] IV. Subcutaneous tumor imaging of MC38-Luc tumor-bearing mice:

[0056] The incubated MC38-Luc cells were subcutaneously injected into the right thigh of the mouse. After 2 weeks, the tumor volume reached 100mm 3 The probe (80μM, 100μL) was injected into the tumor in situ, and the mice were anesthetized and real-time imaging analysis was performed in a near-infrared zone II small animal imager. The imaging results showed that obvious chemiluminescent signals appeared inside the subcutaneous tumor on the right thigh of the tumor-bearing mice, while normal mice had no related signals, indicating that the probe CD-950 can achieve the high expression of H in the tumor of MC38-Luc tumor-bearing mice. 2 S-specific imaging (see Appendix Figure 5 ).

[0057] 5. Imaging of Metformin-Induced Mouse Liver Injury Model:

[0058] First, mice were intraperitoneally injected with different doses of metformin (2mg, 4mg, 6mg) for 7 consecutive days. After the model was successfully established, the probe (200μM, 200μL) was injected through the tail vein, and then the livers of mice treated with different doses of metformin were imaged in real time. The imaging results showed that mice treated with metformin had obvious chemiluminescent signals, and as the dose of metformin increased, the signal intensity became more and more obvious, while no obvious chemiluminescent signals were seen in the livers of normal mice, indicating that the probe CD-950 can achieve high expression of H in liver injury sites. 2 S-specific imaging (see Appendix Figure 6 ).

[0059] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article or device including the elements.

[0060] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A near-infrared second region organic small molecule chemiluminescent probe, characterized in that: The organic small molecule chemiluminescent probe is CD-950, the maximum emission wavelength is at 950 nm, and the structural formula of CD-950 is as follows: 。 2. A near-infrared second region organic small molecule chemiluminescent probe according to claim 1, characterized in that: The organic small molecule chemiluminescent probe uses the m-dinitrophenol structure as the H2S response site; The chemiluminescent donor molecule is Schaap's dioxetane with an emission wavelength of 700 nm, and the fluorophore with a DAD skeleton structure with a fluorescence emission wavelength of 970 nm is used as the energy acceptor. The two are linked together through an amide bond to obtain the chemiluminescent product.

3. The near-infrared second region organic small molecule chemiluminescent probe according to claim 1, characterized in that: The probe can achieve a specific response to H2S in cells.

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