CN116375692B

CN116375692B - Near infrared fluorescent molecular probe for detecting cysteine, preparation method and kit thereof

Info

Publication number: CN116375692B
Application number: CN202310407375.9A
Authority: CN
Inventors: 刘美慧; 曾文彬; 刘祖源; 李石
Original assignee: Hunan Chaoji Testing Technology Co ltd
Current assignee: Hunan Chaoji Testing Technology Co ltd
Priority date: 2023-04-17
Filing date: 2023-04-17
Publication date: 2024-11-08
Anticipated expiration: 2043-04-17
Also published as: CN116375692A

Abstract

A near-infrared fluorescent molecular probe DBCM‑Cys for detecting cysteine, the structural formula is as follows: The near-infrared fluorescent molecular probe described in the present invention can be synthesized in only two steps and is simple to operate. After reacting with cysteine, the fluorescence intensity at 714nm will be significantly enhanced. The fluorescence emission band is in the near-infrared light region, with little background interference, high accuracy and sensitivity, and excellent specificity, and is not interfered by other common amino acids and inorganic salt ions.

Description

Near infrared fluorescent molecular probe for detecting cysteine, preparation method and kit thereof

Technical Field

The invention relates to a near infrared fluorescent molecular probe for detecting cysteine, a preparation method and a kit thereof, belonging to the technical field of small organic molecular fluorescent probes.

Background

Cysteine (Cys) is an important small molecule biological thiol, widely found in biological systems, and associated with a variety of physiological and pathological activities. Studies have shown that Cys plays a key role in maintaining redox balance in homeostasis, and thus, its abnormal levels can lead to the development of a variety of diseases. For example, lack of Cys can lead to slow growth and liver damage, while excessive Cys content can lead to neuropathy such as Alzheimer's disease. Therefore, it is of great importance to achieve detection of cysteine in biological systems. Up to now, ultraviolet-visible absorption spectrophotometry, high performance liquid chromatography tandem mass spectrometry (HPLC-MS) and the like have been developed for detecting Cys. However, these methods are not suitable for Cys detection in living systems such as living cells because the intracellular environment of living cells is very complex and they generally require special equipment and damage and post-mortem treatment of tissues or cells. In contrast, fluorescent molecular probes have high sensitivity, strong specificity, good biocompatibility, and can realize real-time noninvasive detection, and have also been used for Cys detection.

The invention patent application with the application number of CN201710282924.9, named as a fluorescent probe for detecting cysteine, a preparation method and application thereof takes naphthalimide dye as a raw material, designs and synthesizes a naphthalimide-acrylate fluorescent probe, and can emit fluorescence at 669nm when reacting with the cysteine.

In another example, application number CN201710053670.3, entitled "fluorescent probe for identifying cysteine and homocysteine using naphthalimide as parent nucleus" is used for preparing fluorescent probe for identifying cysteine and homocysteine using naphthalimide as parent nucleus, excitation wavelength is 488nm, and emission wavelength is 520nm to 610nm.

The above documents are all fluorescence probes for detecting Cys prepared by taking naphthalimide as a raw material, but the wavelength is not long enough, and the near infrared fluorescence molecular probes (the maximum emission wavelength is 700-900 nm) have better cell penetration capability, less tissue injury, lower background interference and lower cytotoxicity, and are more suitable for detecting Cys in living systems such as cells. Therefore, it is important and significant to develop a near infrared fluorescent probe capable of detecting cysteine.

Disclosure of Invention

Aiming at the problems, the invention provides a near infrared fluorescent molecular probe for detecting cysteine, a preparation method and a kit thereof, wherein the fluorescent molecular probe can be used for specifically detecting the cysteine and has strong anti-interference capability.

The invention adopts the technical means for solving the problems that: a near infrared fluorescent molecular probe DBCM-Cys for detecting cysteine has the following structural formula:

a preparation method of a near infrared fluorescent molecular probe for detecting cysteine comprises the following steps:

(1) Fully dissolving DCM and HBC in acetonitrile solvent, adding piperidine, nitrogen or argon and other inert gases for reflux reaction at 40-60 ℃ until the reaction is finished, removing the reaction solvent under reduced pressure, and purifying by column chromatography to obtain a dark red solid product DBCM; wherein the dosage ratio of DCM to HBC is as follows: 1:0.8-1:1.5 mmol/mmol, the dosage ratio of DCM to solvent is: 1:8-1:15 mmol/mL, the dosage ratio of DCM to piperidine is: 12:1 to 7:1mmol/mmol;

(2) Dissolving DBCM in anhydrous dichloromethane under the protection of inert gas at 0 ℃, dropwise adding triethylamine, finally slowly dropwise adding dichloromethane solution dissolved with acryloyl chloride, stirring at 0 ℃ until the reaction is complete, extracting, combining organic layers, drying, concentrating, and purifying by column chromatography to obtain the target fluorescent probe DBCM-Cys; wherein the dosage ratio of DBCM to anhydrous dichloromethane is as follows: the dosage ratio of DBCM to triethylamine is 1:8-1:20 mmol/mL: 1.5:1-2.5:1 mmol/mL, the concentration of the dichloromethane solution of the acryloyl chloride is as follows: the dosage ratio of DBCM to the acryloyl chloride is 1:4-1:6 mmol/mL: 1:1 to 1:1.5mmol/mmol.

The synthetic route is as follows:

A kit for detecting cysteine, which comprises a reagent stock solution a and a reagent stock solution b, wherein the reagent stock solution a is acetonitrile-PBS buffer solution, the reagent stock solution b is an organic solvent solution of DBCM-Cys fluorescent molecular probes, and the organic solvent is selected from one of dimethyl sulfoxide, N-dimethylformamide and acetonitrile.

Further, the volume ratio of acetonitrile to PBS buffer solution in reagent stock solution a was 3:7, ph=7.4; the concentration of the fluorescent molecular probe in the reagent stock solution b was 1mM.

Further, the volume ratio of the reagent stock solution a and the reagent stock solution b in the kit is 99/1.

The detection principle of the Cys detection kit provided by the invention is as follows:

The method utilizes DCM and naphthalimide fluorophor to construct an Intramolecular Charge Transfer (ICT) system, and the acrylic ester inhibits the ICT effect of the probe to cause no fluorescence emission, but when Cys exists, the acrylic ester bond is broken and replaced by hydroxyl, so that the inhibition effect of the acrylic ester bond disappears, and the probe molecule emits strong fluorescence. The detection mechanism is as follows:

The beneficial effects of the invention are as follows:

1. the near infrared fluorescent molecular probe can be synthesized in only two steps, and is low in cost and simple and convenient to operate.

2. The near infrared fluorescent molecular probe can realize specific detection and high-sensitivity identification of Cys, and Hcy and GSH with similar structures can not interfere the detection.

3. The maximum emission wavelength of the near infrared fluorescent molecular probe is 714nm, and the near infrared fluorescent molecular probe is in a near infrared region, so that the detection of endogenous and exogenous Cys in living cells is facilitated.

Drawings

FIG. 1 is a graph showing the emission spectrum of fluorescence intensity of a fluorescent probe according to Cys concentration in example II;

FIG. 2 is a graph showing the linear relationship between fluorescence intensity and Cys concentration of the fluorescent probe of example II;

FIG. 3 is a graph showing the selectivity of a three fluorescence probe to Cys in the example;

FIG. 4 is a fluorescence confocal image of a fluorescent probe of example four in HeLa cells, wherein a is the image of the cells after 30 minutes of probe treatment; b is an imaging image after 30 minutes of treatment of cells with NEM and then probe.

Detailed Description

The invention is further described below with reference to the accompanying drawings.

Example 1

Synthesis of Compound DBCM

A50 mL round bottom flask was taken, DCM (2- (2-methyl-4H-benzopyran-4-ylidene) malononitrile) (416.44 mg,2 mmol) and HBC (2-butyl-6-hydroxy-1, 3-dioxo-2, 3-dihydro-1H-benzo [ d ] isoquinoline-5-carbaldehyde) (594.62 mg,2 mmol) were added thereto, and acetonitrile 20mL was added thereto to make it fully dissolved, then piperidine (0.2 mmol) was added dropwise to the reaction system, and the reaction system was put under reflux under nitrogen protection at 50 ℃. TLC was used to monitor the progress of the reaction until the end of the reaction, the reaction solvent was removed under reduced pressure, and the product was purified by column chromatography to give DBCM805.37 mg of a dark red solid product in the yield of 82.6％.¹H NMR(400MHz,CDCl₃)δ9.04(s,1H),8.51(d,J＝7.8Hz,1H),8.41(d,J＝7.9Hz,1H),7.80(t,J＝7.8Hz,1H),7.36-7.31(m,2H),7.17(d,J＝7.4Hz,1H),7.03-6.98(m,2H),6.63-6.57(m,2H),5.66(s,1H),3.16-3.13(m,2H),1.79-1.69(m,2H),1.54-1.47(m,2H),0.90(t,J＝7.2Hz,3H).

Synthesis of Compound DBCM-Cys

A25 mL round bottom flask was taken, DBCM (487.52 mg,1 mmol) was added under nitrogen protection at 0deg.C, 10mL of anhydrous dichloromethane was added to make it fully dissolved, triethylamine (0.5 mL) was added dropwise to the reaction system, and finally 5mL of dichloromethane solution in which acryloyl chloride (90.5 mg,1 mmol) was dissolved was slowly added dropwise. Stir overnight at 0 ℃. Extracting with water, saturated NaHCO ₃ solution and saturated NaCl solution respectively, mixing organic layers, drying with anhydrous magnesium sulfate, concentrating, and purifying by column chromatography to obtain target fluorescent probe DBCM-Cys385.59 mg, yield 71.2％.¹H NMR(400MHz,CDCl₃)δ8.51(d,J＝7.6Hz,1H),8.41(d,J＝8.0Hz,1H),7.80(t,J＝7.4Hz,1H),7.37-7.30(m,2H),7.17-7.10(m,2H),6.93(t,J＝7.8Hz,1H),6.63(d,J＝13.8Hz,1H),6.51(s,1H),6.24-6.18(m,2H),5.74(d,J＝6.4Hz,1H),5.66(s,1H),3.18-3.13(m,2H),1.52-1.45(m,4H),0.88(t,J＝7.2Hz,3H).HRMS-ESI(m/z)calc.for C₃₃H₂₃N₃O₅:541.16377;found,[M+H]⁺542.03491.

Example two

The present example is the variation of fluorescence intensity and Cys concentration of the fluorescent probe

Taking 4.82mg of the fluorescent probe prepared in the first embodiment, carefully transferring the probe into a 10mL volumetric flask, adding DMSO at room temperature, shaking the probe uniformly to dissolve the probe completely, and finally fixing the volume to a scale mark to obtain 1mM probe mother solution. 20. Mu.L of the fluorescent probe was dissolved in each of 588. Mu.L and 1372. Mu.L of acetonitrile and PBS buffer solutions, and 20. Mu.L of Cys mother liquor was added thereto at different concentrations, and the total volume of each test was 2mL, so that the concentration of the fluorescent probe in the test system was 10. Mu.M, and the concentration of Cys was 0. Mu.M, 0.5. Mu.M, 1. Mu.M, 2. Mu.M, 5. Mu.M, 10. Mu.M, 15. Mu.M, 20. Mu.M, 30. Mu.M, 50. Mu.M. After incubation for 20min at room temperature for a sufficient response, the fluorescence spectra of the different systems were tested in 10mm cuvettes. The change chart of fluorescence emission spectrum is shown in figure 1. The results showed that the fluorescence intensity of the system at 714nm was gradually increased with increasing Cys concentration. As can be derived from fig. 2, the linear relationship is good in the range of 0-10 μm, the linear equation is y=16.768817x+11.73542, and the linear correlation coefficient is: 0.97492, and a detection Limit (LOD) of 0.17 μm (S/n=3) was calculated, indicating that the fluorescent probe has good sensitivity.

Example III

This example is the selectivity of fluorescent probe response to Cys

20. Mu.L of 1mM probe stock solution was dissolved in 588. Mu.L and 1372. Mu.L of each of acetonitrile and PBS buffer solutions, and then 20. Mu.L of 1mM Cys stock solution and 3mM GSH, hcy, met, leu, ala, phe, trp, tys, glu, naHS, na ₂ S stock solution were respectively added to the detection system, so that the final whole detection system had a probe concentration of 10. Mu.M and Cys concentration of 10. Mu.M and GSH, hcy, met, leu, ala, phe, trp, tys, glu, HS ^-、S^2- concentration of 30. Mu.M. After incubation for 20min at room temperature for full response, fluorescence spectra of different systems are tested in a cuvette of 10mm, relative fluorescence intensity values at 714nm are calculated, corresponding fluorescence intensity at 714nm is taken as an ordinate, and a response histogram of the probe to different substances is obtained, and the result is shown in fig. 3. The results show that the probe has excellent anti-interference performance on the response of Cys and is not influenced by other amino acids and analogues.

Example IV

Investigation of the response of the Probe to Cys in living cells

To the DMEM medium, 10% fetal bovine serum and penicillin (100 units/mL) -streptomycin (100. Mu.g/mL) solution were added, heLa cells were placed in the medium and cultured at 37℃under 5% CO ₂, after 12 hours, the cell culture medium was removed from the wells, and the cells were washed three times with D-Hanks. HeLa cells were then incubated with 20. Mu. M DBCM-Cys and DMEM medium containing 10% fetal bovine serum at 37℃for 30 minutes, then washed twice with PBS and mounted on a microscope stage, and the fluorescence change was observed to see significant fluorescence (FIG. 4 a). Whereas little red fluorescence was observed if HeLa cells were previously treated with N-ethylmaleimide, a widely used thiol blocking agent, and incubated with the probe for another 30 minutes (FIG. 4 b). DBCM-Cys was shown to be useful as a NIR fluorescent molecular probe for imaging Cys in living cells.

The above embodiments are only for illustrating the present invention, not for limiting the present invention, and various changes and modifications may be made by one skilled in the relevant art without departing from the spirit and scope of the present invention, so that all equivalent technical solutions shall fall within the scope of the present invention, which is defined by the claims.

Claims

1. A near-infrared fluorescent molecular probe DBCM-Cys for detecting cysteine, characterized in that: the structural formula is as follows:

2. A method for preparing a near-infrared fluorescent molecular probe for detecting cysteine, characterized in that the steps are as follows:

(1) DCM and HBC are fully dissolved in acetonitrile solvent, piperidine is added, and the reaction is refluxed at 40-60°C under inert gas protection until completion, and DBCM is purified;

(2) Under inert gas protection and at 0 ^° C, DBCM was dissolved in anhydrous dichloromethane, triethylamine was added dropwise, and finally a dichloromethane solution containing acryloyl chloride was slowly added dropwise. The reaction was stirred at 0 ^° C until the reaction was complete. After purification, the target fluorescent probe DBCM-Cys was obtained.

3 . The method for preparing a near-infrared fluorescent molecular probe for detecting cysteine according to claim 2 , wherein the inert gas is nitrogen or argon.

4. The method for preparing a near-infrared fluorescent molecular probe for detecting cysteine according to claim 2, characterized in that: in step (1), the reflux temperature is 50°C.

5. The method for preparing a near-infrared fluorescent molecular probe for detecting cysteine according to claim 2, characterized in that: in step (1), the dosage ratio of DCM to HBC is: 1:0.8~1:1.5 mmol/mmol, the dosage ratio of DCM to solvent is: 1:8~1:15 mmol/mL, and the dosage ratio of DCM to piperidine is: 12:1~7:1 mmol/mmol;

In step (2), the dosage ratio of DBCM to anhydrous dichloromethane is 1:8~1:20 mmol/mL, the dosage ratio of DBCM to triethylamine is 1.5:1~2.5:1 mmol/mL, the concentration of dichloromethane solution of acryloyl chloride is 1:4~1:6 mmol/mL, and the dosage ratio of DBCM to acryloyl chloride is 1:1~1:1.5 mmol/mmol.

6. The method for preparing a near-infrared fluorescent molecular probe for detecting cysteine according to claim 2, characterized in that: in step (1), the dosage ratio of DCM to HBC is 1:1 mmol/mmol, the dosage ratio of DCM to solvent is 1:10 mmol/mL, and the dosage ratio of DCM to piperidine is 10:1 mmol/mmol;

In step (2), the usage ratio of DBCM to anhydrous dichloromethane is 1:10 mmol/mL, the usage ratio of DBCM to triethylamine is 2:1 mmol/mL, the concentration of dichloromethane solution of acryloyl chloride is 1:5 mmol/mL, and the usage ratio of DBCM to acryloyl chloride is 1:1 mmol/mmol.

7. The method for preparing a near-infrared fluorescent molecular probe for detecting cysteine as claimed in claim 2, characterized in that: in step (1), the purification method is to remove the solvent under reduced pressure and purify by column chromatography; in step (2), the purification method is to extract, combine the organic layers, concentrate after drying, and purify by column chromatography.

8. A kit for detecting cysteine, comprising a reagent stock solution a and a reagent stock solution b, characterized in that the reagent stock solution a is an acetonitrile-PBS buffer solution, and the reagent stock solution b is an organic solvent solution of a DBCM-Cys fluorescent molecular probe, wherein the organic solvent is selected from one of dimethyl sulfoxide, N,N-dimethylformamide, and acetonitrile; wherein the DBCM-Cys structural formula is as follows:

9. The kit for detecting cysteine according to claim 8, characterized in that: the volume ratio of acetonitrile and PBS buffer solution in the reagent stock solution a is 3:7, pH=7.4; and the concentration of the fluorescent molecular probe in the reagent stock solution b is 1 mM.

10. The kit for detecting cysteine according to claim 8, characterized in that the volume ratio of the reagent stock solution a to the reagent stock solution b in the kit is 99/1.