阅读说明：本技术 一种基于异硫氰酸酯结构特异性检测半胱氨酸的近红外荧光探针及其制备方法和应用 (Near-infrared fluorescent probe for detecting cysteine based on isothiocyanate structure specificity and preparation method and application thereof ) 是由 葛春坡 任峰 路承彪 于 2021-02-05 设计创作，主要内容包括：本发明公开了一种基于异硫氰酸酯结构特异性检测半胱氨酸的近红外荧光探针及其制备方法和应用,涉及荧光探针技术领域。该近红外荧光探针的结构为：其制备方法包括将第一原料与第二原料反应制得。本申请的近红外荧光探针的异硫氰酸酯基团可以与Cys发生特异性反应的,释放出胺类产物和H-2S,其中,胺类产物具有较强的荧光强度,可以显示近红外荧光探针与Cys发生特异性反应。该近红外荧光探针具有Cys传感特性、灵敏度高、细胞毒性低等优点,TMN-NCS在检测生命系统中的半胱氨酸方面具有潜在的应用前景。(The invention discloses a near-infrared fluorescent probe for detecting cysteine based on isothiocyanate structure specificity and a preparation method and application thereof, and relates to the technical field of fluorescent probes. The structure of the near-infrared fluorescent probe is as follows: the preparation method comprises the step of reacting a first raw material with a second raw material. The isothiocyanate group of the near-infrared fluorescent probe can perform specific reaction with Cys to release amine products and H 2 And S, wherein the amine product has stronger fluorescence intensity and can show that the near-infrared fluorescent probe and Cys have specific reaction. The near-infrared fluorescent probe has Cys sensing characteristicsThe detection kit has the advantages of high performance, high sensitivity, low cytotoxicity and the like, and the TMN-NCS has potential application prospect in the aspect of detecting cysteine in a life system.)

1.A near-infrared fluorescent probe for detecting cysteine based on isothiocyanate structure specificity is characterized in that the structure of the near-infrared fluorescent probe is as follows:

wherein R is₁、R₂、R₃And R₄Independently selected from OCH₃、CH₃H, OH or N (CH)₃)₂。

2. The method for preparing the near-infrared fluorescent probe for detecting cysteine based on isothiocyanate structure specificity according to claim 1, wherein the probe is prepared by reacting a first raw material and a second raw material, and the first raw material has a structural formula shown in the specificationThe structural formula of the second raw material is as follows:wherein R is₁、R₂、R₃And R₄Independently selected from OCH₃、CH₃H, OH or N (CH)₃)₂。

3. The method for preparing a near-infrared fluorescent probe for the structure-specific detection of cysteine based on isothiocyanate according to claim 2, wherein the molar ratio of the first raw material to the second raw material is 1: 1-2.

4. The method for preparing a near-infrared fluorescent probe for the structure-specific detection of cysteine based on isothiocyanate according to claim 2, wherein the first raw material and the second raw material are reacted in anhydrous DMF containing a catalyst.

5. The method for preparing the near-infrared fluorescent probe for detecting cysteine based on isothiocyanate structure specificity according to claim 4, wherein the first raw material is dissolved in the anhydrous DMF containing the catalyst, the second raw material is added dropwise, and the reaction is carried out for 2-3h to obtain a reaction mixture; pouring the reaction mixture into water, adjusting the pH to 7-8, filtering and collecting a solid product, and purifying the solid product;

preferably, the catalyst comprises one or more of triethylamine, diisopropylethylamine, piperidine and pyridine;

preferably, one or more of dilute hydrochloric acid, dilute nitric acid, dilute sulfuric acid, sodium hydroxide and sodium bicarbonate is used to adjust the pH.

6. The method for preparing the near-infrared fluorescent probe for the structure-specific detection of cysteine based on isothiocyanate according to claim 5, wherein the second raw material is added under a protective atmosphere;

preferably, the protective atmosphere comprises at least one of nitrogen, carbon dioxide, hydrogen, helium and argon.

7. The use of the near-infrared fluorescent probe for the structure-specific detection of cysteine based on isothiocyanate according to claim 1 for the detection of cysteine.

8. The use according to claim 7, wherein the isothiocyanate group in the near-infrared fluorescent probe serves as a reaction site for cysteine.

9. The use according to claim 7, wherein the excitation wavelength range required for detection is 400-500nm and the fluorescence emission wavelength range is 575-780 nm.

10. The use according to claim 7, wherein the near-infrared fluorescent probe is used for fluorescent imaging or concentration detection of intracellular cysteines, non-disease diagnosis and therapeutic purposes.

Technical Field

The invention relates to the technical field of fluorescent probes, in particular to a near-infrared fluorescent probe for detecting cysteine based on isothiocyanate structure specificity and a preparation method and application thereof.

Background

Cysteine (Cys) is an amino acid containing a thiol structure, is a reducing agent or an antioxidant, and has a good scavenging effect on free radicals. Cys plays an important role in many pathological events and biological processes, such as participation in protein synthesis, and exerts a cytoprotective effect by regulating Reactive Oxygen Species (ROS) homeostasis. In addition, Cys concentration abnormalities are also a manifestation of certain diseases in humans, and Cys concentration has been used as a diagnostic indicator for various diseases. Therefore, it is of great physiological significance to develop a simple and effective strategy for selectively detecting Cys in living systems.

Over the past two decades, a number of methods for detecting cysteine have been established. Among the numerous methods, the use of small molecule probe fluorescence imaging has proven to be an indispensable or desirable approach. This is because the fluorescent probe has obvious practical advantages of simple operation, good biocompatibility, high sensitivity, low cost, and the like. Currently, construction of fluorescent probes with Cys-specific recognition sites has been used to design synthetic selective detection of Cys. For example, cyclization reaction of acrylate structure based on Michael addition, cyclization reaction with aldehyde, disulfide exchange reaction, nucleophilic substitution reaction, and the like. However, homocysteine (Hcy) and Glutathione (GSH) have similar characteristics in reactivity and structure to Cys, and there are some drawbacks to using these probes to selectively analyze Cys in Hcy and GSH. In order to avoid interference of Hcy and GSH, a new method for selectively identifying Cys needs to be established.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a near-infrared fluorescent probe for detecting cysteine based on isothiocyanate structure specificity, which has the advantages of good Cys sensing characteristic, high sensitivity and low cytotoxicity.

The second purpose of the invention is to provide a preparation method of the near-infrared fluorescent probe for detecting cysteine based on isothiocyanate structure specificity, and the preparation method is simple and easy to implement.

The third purpose of the invention is to provide an application of the near-infrared fluorescent probe for detecting cysteine based on isothiocyanate structure specificity in cysteine detection.

The invention is realized by the following steps:

in a first aspect, the present invention provides a near-infrared fluorescent probe for detecting cysteine based on isothiocyanate structure specificity, wherein the structure of the near-infrared fluorescent probe is:

wherein R is₁、R₂、R₃And R₄Independently selected from OCH₃、CH₃H, OH or N (CH)₃)₂。

In a second aspect, the present invention provides a method for preparing a near-infrared fluorescent probe for detecting cysteine based on isothiocyanate structure specificity, the near-infrared fluorescent probe being prepared by reacting a first raw material with a second raw material, the first raw material having a formula ofThe structural formula of the second raw material is as follows:wherein R is₁、R₂、R₃And R₄Independently selected from OCH₃、CH₃H, OH or N (CH)₃)₂。

In an alternative embodiment, the molar ratio of the first feedstock to the second feedstock is from 1:1 to 2.

In an alternative embodiment, the first starting material is reacted with the second starting material in anhydrous DMF containing a catalyst.

In an alternative embodiment, the first raw material is dissolved in the anhydrous DMF containing triethylamine, the second raw material is added dropwise, and the reaction is carried out for 2-3h to obtain a reaction mixture; pouring the reaction mixture into water, adjusting the pH to 7-8, filtering and collecting a solid product, and purifying the solid product;

preferably, the catalyst comprises one or more of triethylamine, diisopropylethylamine, piperidine and pyridine;

preferably, one or more of dilute hydrochloric acid, dilute nitric acid, dilute sulfuric acid, sodium hydroxide and sodium bicarbonate is used to adjust the pH.

In other embodiments herein, the second feedstock is added under a protective atmosphere;

preferably, the protective atmosphere comprises at least one of nitrogen, carbon dioxide, hydrogen, helium and argon.

In a third aspect, the present invention provides the use of the near-infrared fluorescent probe for the structure-specific detection of cysteine based on isothiocyanate described in the previous embodiments in the detection of cysteine.

In an alternative embodiment, the isothiocyanate group in the near-infrared fluorescent probe serves as a reaction site for cysteine.

In an alternative embodiment, the excitation wavelength range required for detection is 400-500nm, and the fluorescence emission wavelength range is 575-780 nm.

In alternative embodiments, the near-infrared fluorescent probes are used for fluorescent imaging or concentration detection of intracellular cysteines, non-disease diagnostic and therapeutic purposes.

The invention has the following beneficial effects:

the application provides a near-infrared fluorescent probe for detecting cysteine based on isothiocyanate structure specificity, and an isothiocyanate group of the near-infrared fluorescent probe can perform specific reaction with Cys to release an amine product and H₂And S, wherein the amine product has stronger fluorescence intensity and can show that the near-infrared fluorescent probe and Cys have specific reaction. Experiments show that the near-infrared fluorescent probe provided by the application has the advantages of Cys sensing characteristic, high sensitivity, low cytotoxicity and the like, and the TMN-NCS has a potential application prospect in the aspect of detecting cysteine in a life system. In addition, the isothiocyanate-based conjugates provided hereinThe near-infrared fluorescent probe for detecting cysteine by structure specificity has the advantages of simple preparation method, easy implementation of operation conditions and suitability for popularization.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 shows a near-infrared fluorescent probe for the structure-specific detection of cysteine based on isothiocyanate, provided in example 1 of the present application¹A HNMR map;

FIG. 2 shows a near-infrared fluorescent probe for the structure-specific detection of cysteine based on isothiocyanate, provided in example 1 of the present application¹³A CNMR map;

FIG. 3 is a graph showing changes in absorption spectrum and fluorescence spectrum before and after the reaction between the near-infrared fluorescent probe for detecting cysteine based on isothiocyanate structure specificity and Cys provided in example 1 of the present application;

FIG. 4 is a fluorescence signal enhancement spectrum of the near-infrared fluorescent probe for detecting cysteine based on isothiocyanate structure specificity after reacting with Cys of different concentrations, provided in example 1 of the present application;

FIG. 5 is a diagram showing the selectivity of a near-infrared fluorescent probe for the structure-specific detection of cysteine based on isothiocyanate provided in example 1 of the present application;

FIG. 6 is an anti-interference diagram of a near-infrared fluorescent probe for the structure-specific detection of cysteine based on isothiocyanate provided in example 1 of the present application;

FIG. 7 is a schematic diagram showing the survival rate of HepG2 cells after incubation of different concentrations of isothiocyanate structure-specific cysteine-based near infrared fluorescent probes provided in example 1 of the present application in HepG2 cells for 24 h.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The application provides a near-infrared fluorescence probe based on isothiocyanate structure specificity detects cysteine, its structure is:

wherein R is₁、R₂、R₃And R₄Independently selected from OCH₃、CH₃H, OH or N (CH)₃)₂。

The structure of the near-infrared fluorescent probe provided by the application has an isothiocyanate group, and the isothiocyanate is an important antifungal and anticancer compound and is widely distributed in cruciferous vegetables. The research of the inventor finds that the isothiocyanate group can specifically react with Cys, and the specific response mechanism (by R)₁、R₂、R₃And R₄Compounds in which all substituents are H) are as follows:

the mechanism shows that the isothiocyanate group can react with Cys specifically to release amine products and H₂And S. The amine product has stronger fluorescence intensity and can show that the near-infrared fluorescent probe and Cys have specific reaction.

The near-infrared fluorescent probe provided by the application is a novel fluorescent probe with red light emission wavelength. The fluorescent material has excellent selectivity on Cys, obvious fluorescence intensity after reaction with Cys, good anti-interference performance, sensing characteristic, high sensitivity, low cytotoxicity and the like, and the TMN-NCS has potential application prospect in the aspect of detecting cysteine in a life system.

Compared with other compounds with NCS structures, the near-infrared fluorescent probe with the structure has the advantages of simple synthesis, high yield and the like, and is more suitable for industrial production and application.

Further, the application also provides a preparation method of the near-infrared fluorescent probe, and the synthetic route is as follows:

wherein R is₁、R₂、R₃And R₄Independently selected from OCH₃、CH₃H, OH or N (CH)₃)₂。

The specific operation steps are as follows:

(1) preparing the first feedstock

With R of the first starting material₁、R₂、R₃And R₄The compound is exemplified by the case where all substituents are H, and the structural formula thereof is:its name TMN-NH is defined herein₂Wherein the name: (E) -2- (3- (4-aminostyryl) -5, 5-dimethylcyclohex-2-en-1-ylidene) malononitrile (prepared according to the literature: Tetrahedron Letters,2020,61, 151963).

(2) Preparation of near Infrared fluorescent Probe (TMN-NCS)

Reacting TMN-NH₂And reacting with a second raw material (1, 1' -thiocarbonyl diimidazole) to prepare the near-infrared fluorescent probe (TMN-NCS). TMN-NH₂With 1, 1' -thiocarbonyldiimidazole in anhydrous DMF with catalyst. Specifically, TMN-NH is first introduced₂Dissolving in anhydrous DMF containing a catalyst, dropwise adding 1, 1' -thiocarbonyl diimidazole under a protective atmosphere, and reacting for 2-3h to obtain a reaction mixture; pouring the reaction mixture into water to quench the reaction, adjusting pH to 7-8, filtering and collecting the solid product, and performing column chromatography on the solid productAnd (5) purifying.

In this application, TMN-NH₂The molar ratio of the compound to 1, 1' -thiocarbonyldiimidazole is 1: 1-2. TMN-NH can be made available by dropwise addition of 1, 1' -thiocarbonyldiimidazole₂Fully reacts with the 1,1 ' -thiocarbonyl diimidazole, and after the reaction is completed, the redundant 1,1 ' -thiocarbonyl diimidazole can be dissolved in water and decomposed by adding water, so that the influence of the 1,1 ' -thiocarbonyl diimidazole on the final product is avoided.

Preferably, the catalyst includes, but is not limited to, one or more of triethylamine, diisopropylethylamine, piperidine, and pyridine, and the pH is adjusted using one or more of dilute hydrochloric acid, dilute nitric acid, dilute sulfuric acid, sodium hydroxide, and sodium bicarbonate. The protective atmosphere includes, but is not limited to, at least one of nitrogen, carbon dioxide, hydrogen, helium, and argon. In the application, the protective atmosphere is adopted to be beneficial to avoiding oxidation in the reaction process. The preparation method provided by the application is simple and easy to implement.

Furthermore, the near-infrared fluorescent probe for detecting cysteine based on isothiocyanate structure specificity can be widely applied to cysteine detection.

In particular, the near-infrared fluorescent probe can be applied to the fluorescent imaging or concentration detection of cysteine in cells, and is not used for disease diagnosis and treatment purposes.

Wherein, the isothiocyanate group in the near-infrared fluorescent probe is used as a reaction site of cysteine to perform atopic detection on Cys, the required excitation wavelength range during detection is 400-500nm, and the fluorescence emission wavelength range is 575-780 nm.

In addition, applicants speculate that compounds 1-5 having the following structure may also be useful as near-infrared fluorescent probes:

the features and properties of the present invention are described in further detail below with reference to examples.

Example 1

The embodiment provides a near-infrared fluorescent probe for detecting cysteine based on isothiocyanate structure specificity, which has a structural formula as follows:

the preparation method comprises the following steps:

in a round-bottomed flask, 0.29g of TMN-NH was charged₂Dissolved in 15mL of anhydrous DMF containing triethylamine. After dissolving the solid by stirring, 0.22g of a solution of 1, 1' -thiocarbonyldiimidazole dissolved in dry DMF was added dropwise under a nitrogen atmosphere. After 2 hours, the reaction mixture was poured into deionized water and the pH was adjusted to 7-8 with dilute HCl. The solid product was collected by filtration and further purified by column chromatography to give TMN-NCS0.28 g (yield 87%) as a yellow powder.

Referring to fig. 1 and 2, the structure is characterized: 1H NMR (400MHz, Chloroform-d) δ 7.49(d, J ═ 8.6Hz,2H),7.24(d, J ═ 8.6Hz,2H),6.99(d, J ═ 16Hz,1H),6.98(d, J ═ 16Hz,1H),6.86(s,1H),2.61(s,2H),2.46(s,2H),1.09(s, 6H).

13C NMR(100MHz,Chloroform-d)δ169.01,153.10,135.10,134.65,132.08,130.19,128.57,126.40,124.25,113.32,112.56,79.38,42.94,39.15,32.04,28.01。

Experimental example 1

The implementation method comprises the following steps: at room temperature, 3mL of a PBS buffer solution (10mM, pH 7.4; v/v) containing 30% DMSO was placed in a cuvette, and the probes TMN-NCS and Cys were added to give final concentrations of 10. mu.M and 100. mu.M, respectively. Spectral data were measured before and after reaction with Cys, respectively. In the fluorescence spectrum measurement, the excitation wavelength is 450nm, and the emission wavelength of 575-780nm is collected (see FIG. 3).

As can be seen from fig. 3, before and after the near-infrared fluorescent probe reacts with Cys, the wavelength at which absorbance is generated changes, and meanwhile, before the near-infrared fluorescent probe reacts with Cys, there is no significant fluorescence intensity, and after the near-infrared fluorescent probe reacts with Cys, there is significant fluorescence intensity, which fully illustrates that the near-infrared fluorescent probe provided by the present application can react with Cys and exhibit fluorescence.

Furthermore, in this experimental example, the spectrum data of the probe TMN-NCS (10. mu.M) after the action with Cys (0, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 200. mu.M) at various concentrations was examined.

As can be seen in FIG. 4, the intensity of the increase in the fluorescence signal between probe TMN-NCS and Cys increased with increasing concentration of Cys.

Experimental example 2

The selectivity is an important condition for determining the performance of the fluorescent probe, and the application also aims at the selectivity experiment of the near-infrared fluorescent probe TMN-NCS on Cys and other common substances.

The experimental method comprises the following steps: 3mL of a PBS buffer solution (10mM, pH 7.4; v/v) containing 30% DMSO was placed in a cuvette at room temperature, a probe TMN-NCS was added to give a final concentration of 10. mu.M, and then each of analytes (1. Ala; 2. Arg; 3. Asn; 4. Asp; 5. Gln; 6. Gly; 7. Glu; 8. Ile; 9. His; 10. Leu; 11. Met; 12. Lys; 13. Phe; 14. Pro; 15. Ser; 16. Thr; 17. Trp; 18. Tyr; 19. Val; 20. KI; 21.MgSO 2. mu.M) was added to give a final concentration of 500. mu.M₄；22.NaBr；23.NaCl；24.NaF；25.CaCl₂(ii) a GSH; hcy; NaHS; cys 29). Wherein Cys is added simultaneously with addition of analyte 1-28 in the detection of anti-interference. Collecting the emission wavelength of 575-780nm with the excitation wavelength of 450 nm.

As can be seen from the selectivity chart of FIG. 5, the near-infrared fluorescent probe TMN-NCS provided by the present application has significantly higher selectivity for Cys than other analytes. As can be seen from the anti-interference graph of FIG. 6, the near-infrared fluorescent probe TMN-NCS provided by the application can effectively avoid the interference of other analysis substances, and has stronger selectivity.

Experimental example 3

Cytotoxicity test

After incubation of different concentrations of TMN-NCS in HepG2 cells for 24 hours, the survival rate of HepG2 cells was measured by MTT method.

As can be seen from FIG. 7, after incubation for 24h, the survival rate of HepG2 cells is high, which indicates that the near-infrared fluorescent probe TMN-NCS provided by the application has low cytotoxicity and has potential application prospect in the aspect of detecting cysteine in a living system.

In summary, the near-infrared fluorescent probe for detecting cysteine based on isothiocyanate structure specificity is provided, and the isothiocyanate group of the near-infrared fluorescent probe can perform specific reaction with Cys to release amine products and H₂And S, wherein the amine product has stronger fluorescence intensity and can show that the near-infrared fluorescent probe and Cys have specific reaction. Experiments show that the near-infrared fluorescent probe provided by the application has the advantages of Cys sensing characteristic, high sensitivity, low cytotoxicity and the like, and the TMN-NCS has a potential application prospect in the aspect of detecting cysteine in a life system. In addition, the preparation method of the near-infrared fluorescent probe for detecting cysteine based on isothiocyanate structure specificity is simple, the operation conditions are easy to implement, and the near-infrared fluorescent probe is suitable for popularization.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.