Preparation process of a GSH ratio fluorescent probe

Abstract

The application discloses a preparation process of a GSH ratio fluorescent probe, and specifically comprises the following steps: (1) raw materials 3-[3-(4-fluorophenyl)-1-isopropyl-1H-indole-2-yl]-propenal and dehydroacetic acid are placed in a 25 mL round-bottom flask, anhydrous DMSO is added as a solvent, and the raw materials are completely dissolved; (2) a catalytic amount of piperidine is added dropwise as a catalyst, and the reaction is carried out under N2 protection at 45±2 DEG C for 24 hours; after the reaction is completed, the color of the solution changes from the original blood red to dark red; (3) after cooling, dichloromethane and water are used for extraction, and finally, a dichloromethane / petroleum ether mixture is used as a developing agent to obtain a red crystal, which is used as a probe molecule 1 of the GSH ratio fluorescent probe. The application relates to the technical field of biological medicine, and specifically provides a preparation process of a GSH ratio fluorescent probe.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a preparation process for a fluorescent probe for detecting GSH ratios. Background Technology

[0002] Glutathione (GSH) is a tripeptide containing a γ-amide bond and a thiol group, composed of glutamic acid, cysteine, and glycine, and is present in almost every cell of the body. Glutathione helps maintain normal immune system function and has antioxidant and detoxification effects. GSH plays a vital role in human health, and abnormal GSH levels are directly related to various diseases. Therefore, many researchers are interested in GSH level detection, leading to the development of numerous fluorescent GSH probes and thus contributing to the advancement of this field.

[0003] Currently, publicly available GSH-specific fluorescent probes primarily utilize the strong nucleophilicity of the thiol group (-SH) in the GSH structure as their design starting point. The probe's recognition group is attacked by the thiol structure in GSH, undergoing a nucleophilic reaction or bond cleavage reaction, leading to changes in the fluorescence signal of the probe's surrounding environment, altering its color and fluorescence, thereby achieving recognition. Based on the different groups at the reaction recognition site, these probes can be mainly classified into oxygen / thioether bond types, sulfonate / sulfonamide types, disulfide bond types, carbon-halogen bond types, selenium-nitrogen bond types, and unsaturated double bond types. In modern technology, although chemical sensors possess extremely high sensitivity and selectivity, they still struggle to distinguish between the three thiols (glutathione (GSH), cysteine ​​(Cys), and homocysteine ​​(Hcy)) due to their extremely similar structures. Therefore, synthesizing selectively recognizing biological thiols is essential. Summary of the Invention

[0004] To address the aforementioned shortcomings, this invention provides a preparation process for a GSH ratio fluorescent probe with mild reaction conditions, simple operation steps and post-processing, and double bond detection. This fluorescent probe utilizes the strong nucleophilicity of the thiol group in GSH, which readily undergoes a Michael nucleophilic addition reaction with the unsaturated C / C double bond. The thiol group adds to the double bond, disrupting the conjugated system in the molecule and causing a change in the fluorescence signal, thereby achieving the recognition effect.

[0005] This invention provides the following technical solution: The preparation process of a ratiometric fluorescent probe for detecting GSH proposed in this invention specifically includes the following steps:

[0006] (1) Place the raw materials 3-[3-(4-fluorophenyl)-1-isopropyl-1H-indol-2-yl]-propenal and dehydroacetic acid in a 25 mL round-bottom flask, add anhydrous DMSO as a solvent and completely dissolve the raw materials;

[0007] (2) Add a catalytic amount of piperidine as a catalyst, and react for 24 hours under N2 protection at 45±2℃. After the reaction is complete, the color of the solution changes from the original blood red to dark red.

[0008] (3) After cooling, the sample was extracted with dichloromethane and water. Finally, a dichloromethane / petroleum ether mixture was used as the developing solvent to obtain red crystals, which were used as probe molecule 1 for detecting GSH ratio fluorescent probe.

[0009] Furthermore, the chemical formula for the preparation and synthesis of the probe molecule 1 is as follows:

[0010] .

[0011] Furthermore, in step (3), the volume ratio of dichloromethane to petroleum ether in the dichloromethane / petroleum ether mixture is 1:1.

[0012] The beneficial effects achieved by the present invention using the above structure are as follows: The preparation process of the GSH ratio fluorescent probe proposed in this invention has mild reaction conditions, simple operation steps and post-processing, and contains double bond detection function. Because the thiol group in GSH has strong nucleophilicity, it is easy to undergo Michael nucleophilic addition reaction with unsaturated C / C double bond. The thiol group adds to the double bond, which destroys the conjugated system in the molecule, thereby causing a change in the fluorescence signal, thus achieving the recognition effect. Attached Figure Description

[0013] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0014] Figure 1 The UV-Vis absorption spectrum of probe molecule 1 after reacting with various amino acids in an ethanol solution of the probe molecule 1, which is part of the preparation process of the GSH ratio fluorescent probe proposed in this invention.

[0015] Figure 2 The fluorescence spectrum of probe molecule 1 after reacting with various amino acids in an ethanol solution of the preparation process of the GSH ratio fluorescent probe proposed in this invention.

[0016] Figure 3 The probe molecule 1 is a preparation process for a GSH ratiometric fluorescent probe proposed in this invention. 1 HNMR spectrum;

[0017] Figure 4 The probe molecule 1 is a preparation process for a GSH ratiometric fluorescent probe proposed in this invention. 13CNMR spectrum. Detailed Implementation

[0018] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0019] A preparation process for a ratiometric fluorescent probe for detecting GSH specifically includes the following steps:

[0020] (1) Place the raw materials 3-[3-(4-fluorophenyl)-1-isopropyl-1H-indol-2-yl]-propenal and dehydroacetic acid in a 25 mL round-bottom flask, add anhydrous DMSO as a solvent and completely dissolve the raw materials;

[0021] (2) Add a catalytic amount of piperidine as a catalyst, and react for 24 hours under N2 protection at 45±2℃. After the reaction is complete, the color of the solution changes from the original blood red to dark red.

[0022] (3) After cooling, the mixture is extracted with dichloromethane and water. Finally, a dichloromethane / petroleum ether mixture is used as the developing solvent to obtain red crystals. The volume ratio of dichloromethane to petroleum ether is 1:1. The red crystals are used as probe molecule 1 for detecting GSH ratio fluorescent probes.

[0023] The chemical formula for the preparation and synthesis of probe molecule 1 is as follows:

[0024] .

[0025] To verify whether probe molecule 1 can selectively recognize GSH / Cys / Hcy, probe molecule 1 was completely dissolved in ethanol solution to prepare an ethanol solution of probe molecule 1 (2×10⁻⁶). -5 mol / L), then prepare thiols (GSH, Hcy, and Cys) and other L-amino acids (Iso, Ser, Arg, Gln, His, Leu, Lys, Asp, Val, Met, Thr, Pro, α-Ala) (2 × 10⁻⁶ mol / L), and then prepare thiols (GSH, Hcy, and Cys) and other L-amino acids (Iso, Ser, Arg, Gln, His, Leu, Lys, Asp, Val, Met, Thr, Pro, α-Ala) (2 × 10⁻⁶ mol / L). -2 A mol / L aqueous solution was prepared and added dropwise to the solution of probe molecule 1.

[0026] like Figure 1As shown, the ethanol solution of probe molecule 1 has absorption at 415 nm. When 8 equivalents of GSH and other amino acids are added, a new absorption peak appears at 455 nm and is more obvious only after the addition of GSH. The appearance of this new absorption peak fully demonstrates that the addition of GSH leads to changes in UV-Vis absorption.

[0027] However, after adding equal amounts of two thiols (Hcy, Cys) and other amino acids and reacting for 1 minute, the color and UV-Vis absorption intensity of the solutions except for those with Asp and Thr did not change significantly. Although the UV-Vis absorption of Asp and Thr changed, the UV-Vis absorption of these two amino acids was very different from that of GSH, so they could still be distinguished.

[0028] like Figure 2 As shown, the fluorescence intensity of probe molecule 1 with added GSH and other amino acids was tested simultaneously. The maximum fluorescence emission of the ethanol solution of probe molecule 1 was at 567 nm in the fluorescence spectrum. After adding GSH, the fluorescence intensity at 567 nm decreased significantly, while the fluorescence intensity at 567 nm increased after adding two other thiols and other amino acids.

[0029] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, material, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, material, or apparatus.

[0030] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A preparation process for a ratiometric fluorescent probe for detecting GSH, characterized in that, Specifically, the following steps are included: (1) Place the raw materials 3-[3-(4-fluorophenyl)-1-isopropyl-1H-indol-2-yl]-propenal and dehydroacetic acid in a 25 mL round-bottom flask, add anhydrous DMSO as a solvent and completely dissolve the raw materials; (2) Add a catalytic amount of piperidine as a catalyst, and react for 24 hours under N2 protection at 45±2℃. After the reaction is complete, the color of the solution changes from the original blood red to dark red. (3) After cooling, the sample was extracted with dichloromethane and water, and finally red crystals were obtained by using a dichloromethane / petroleum ether mixture as the developing solvent. The red crystals were used as probe molecule 1 for detecting GSH ratio fluorescent probe. The structure of probe molecule 1 is as follows: 。 2. The preparation process of the GSH ratio fluorescent probe according to claim 1, characterized in that, The chemical formula for the preparation and synthesis of probe molecule 1 is as follows: 。 3. The preparation process of the GSH ratio fluorescent probe according to claim 1, characterized in that, In step (3), the volume ratio of dichloromethane to petroleum ether in the dichloromethane / petroleum ether mixture is 1:1.

Images