Fluorescent probe for identifying cysteine and glutathione as well as preparation method and application thereof

A technology of glutathione and cysteine, applied in the field of chemical analysis and detection, can solve the problem of not having the function of targeting lysosomes, and achieve the effects of high sensitivity, mild reaction conditions and simple synthesis route

Active Publication Date: 2018-11-02

南宁师范大学

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AI-Extracted Technical Summary

Problems solved by technology

[0004] At present, most of the probes used to detect and distinguish cysteine and glutathione do not have the function of targeting lyso...

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Method used

1, as can be seen from Fig. 7 and Fig. 8, add Tyr, Val, Gly, Ala, Asp, Arg, Iso, Lys, Met, His, Phe, Thr, Ser, Pro, Glu, KCl in solution , CaCl2, MgCl2, ZnCl2, NaCl, H2S, and H2O2, compared with only adding fluorescent probes in the solution, the fluorescence intensity did not change, while adding two kinds of Cys and GHS amino acids respectively in the solution, the fluorescent p...

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Abstract

The invention discloses a fluorescent probe for identifying cysteine and glutathione. The structure of the probe is a chemical formula: the formula is shown in the description. The preparation methodof the fluorescent probe for identifying the cysteine and the glutathione comprises the following steps: S1, performing reaction by taking 3-carboxyl-4-chlorine-7-diethylamine coumarin and 3-hydroxyl-N-(2-ethyl morpholine)benzamide as raw materials to obtain an intermediate; and S2, performing reaction by taking the intermediate and p-nitrophenol as raw materials to obtain a probe. The fluorescentprobe can identify the cysteine and the glutathione from various kinds of amino acid and some common substances, has a lysosome targeted function at the same time, can directionally detect the cysteine and the glutathione in the lysosome, has good selectivity and high sensitivity on the cysteine and the glutathione, and has a good application prospect in detection and analysis of the cysteine andthe glutathione in environments or biological samples.

Application Domain

Organic chemistryFluorescence/phosphorescence +1

Technology Topic

FluorescenceAmino acid +10

Image

Examples

Experimental program(3)

Example Embodiment

[0050]

[0051] Preparation of intermediates

[0052] Using 3-carboxy-4-chloro-7-diethylaminocoumarin and 3-hydroxy-N-(2-ethylmorpholine)benzamide as raw materials to react to obtain intermediates, the specific process includes:

[0053] Step 1: Add dry dichloromethane (5mL) to 3-carboxy-4-chloro-7-diethylaminocoumarin (295mg, 1mmol), stir to dissolve, and then add oxalyl chloride (0.9mL, 10mmol) in sequence , Anhydrous DMF (5μL), react at room temperature under argon protection for 2.5 hours, distill under reduced pressure to remove the solvent, and then add anhydrous dichloromethane to dissolve (5mL) to obtain the first reaction solution;

[0054] Step 2. Add 3-hydroxy-N-(2-ethylmorpholine)benzamide (1mmol), anhydrous triethylamine (4.4mL, 30mmol) to anhydrous dichloromethane (5mL) and stir to dissolve to obtain the Two reaction liquid;

[0055] Step 3. Add the first reaction solution to the second reaction solution at °C, react for 40 minutes in an ice-water bath, distill off the solvent under reduced pressure, and pass column chromatography (MeOH/EA, 50/1v/v) to obtain Light yellow solid, which is the intermediate, the yield is 279 mg, the yield is 50%, 1 The HNMR spectrum is as figure 1 As shown, 13 The CNMR spectrum is as figure 2 As shown, the mass spectrum is as image 3 Shown.

Example Embodiment

[0056]

[0057] Preparation of fluorescent probe:

[0058] The probe is obtained by reacting the intermediate and p-nitrophenol as raw materials. The specific process for obtaining the probe is as follows:

[0059] Add anhydrous acetonitrile (7mLl) to the intermediate (577mg, 1mmol) and p-nitrophenol (139mg, 1mmol) and stir to dissolve (specifically, the intermediate and nitrophenol are added to the reactor first, and then added to the reactor Anhydrous acetonitrile), then add triethylamine (5μL, 1mmol), under the protection of argon, reflux and stir for 1.5h, distill off the solvent under reduced pressure, and obtain a light yellow solid by column chromatography. The light yellow solid is the probe ( probe), yield 577mg, 85%, 1 The HNMR spectrum is as Figure 4 As shown, 13 The CNMR spectrum is as Figure 5 As shown, the mass spectrum is as Image 6 Shown.

Example Embodiment

[0060]

[0061] Application of fluorescent probe

[0062] Dissolve the fluorescent probe in the buffer solution (V DMSO /V PBS = 2/8, Ph = 7.4), and mixed into multiple groups 1.0×10 - 5 mol/L solution, and then add Cys, GSH, Tyr, Val, Gly, Ala, Asp, Arg, Iso, Lys, Met, His, Phe, Thr, Ser, Pro, Glu to multiple solutions one by one. , KCl, CaCl 2 , MgCl 2 , ZnCl 2 , NaCl, H 2 S, and H 2 O 2 , And test the fluorescence intensity of each group of solutions, the test results are as follows Figure 7 with Picture 8 Shown

[0063] Dissolve the fluorescent probe in the buffer solution (V DMSO /V PBS = 2/8, Ph = 7.4), and mixed into multiple groups 1.0×10 - 5 mol/L solution, and then add cysteine (Cys) of different masses to multiple groups of solutions one by one to prepare a solution with a cysteine concentration of 0-300μM, and finally test the fluorescence intensity respectively, the results are as follows Picture 9 As shown, the arrow in the figure indicates that the concentration of cysteine is gradually increasing, and the linear relationship between the concentration of cysteine (Cys) and the fluorescence intensity is obtained, such as Picture 10 Shown

[0064] Dissolve the fluorescent probe in the buffer solution (V DMSO /V PBS = 2/8, Ph = 7.4), and mixed into multiple groups 1.0×10 - 5 mol/L solution, and then add glutathione (GHS) of different quality to multiple groups of solutions one by one to prepare a solution with a glutathione concentration of 0-500μM, and finally test its fluorescence intensity. The results are as follows Picture 11 As shown, Picture 11 The middle arrow points to indicate that the concentration of glutathione gradually increases, and the linear relationship between the concentration of glutathione and the fluorescence intensity is obtained, such as Picture 12 Shown

[0065] Dissolve the fluorescent probe in the buffer solution (V DMSO /V PBS = 2/8, Ph = 7.4), and formulated to 1.0×10 -5 mol/L solution, then add cysteine (Cys) to the solution to prepare a solution with a cysteine concentration of 300μM, and finally test the fluorescence intensity at different time points, the results are as follows Figure 13 And get the relationship between fluorescence intensity and time, such as Figure 14 Shown

[0066] Dissolve the fluorescent probe in the buffer solution (V DMSO /V PBS = 2/8, Ph = 7.4), and formulated to 1.0×10 -5 mol/L solution, then add glutathione (GHS) to the solution to prepare a solution with a glutathione concentration of 500μM, and finally test the fluorescence intensity at different time points, the results are as follows Figure 15 And get the relationship between fluorescence intensity and time, such as Figure 16 Shown

[0067] Dissolve the fluorescent probes in PBS buffer solutions of different Ph and prepare them to 1.0×10 -5 mol/L solution, test the fluorescence intensity of fluorescent probes under different Ph conditions, then add cysteine (Cys) to the solution and prepare a solution with a cysteine concentration of 300μM, and finally test the fluorescence intensity with PH changes such as Figure 17 Shown

[0068] Dissolve the fluorescent probes in PBS buffer solutions of different Ph and prepare them to 1.0×10 -5 mol/L solution, test the fluorescence intensity of the fluorescent probe under different Ph conditions, then add glutathione (GHS) to the solution, and prepare a solution with a glutathione concentration of 500μM, and finally test the fluorescence intensity with Ph changes like Figure 18 Shown.

[0069] the result shows:

[0070] 1. By Figure 7 with Picture 8 It can be seen that adding Tyr, Val, Gly, Ala, Asp, Arg, Iso, Lys, Met, His, Phe, Thr, Ser, Pro, Glu, KCl, CaCl to the solution 2 , MgCl 2 , ZnCl 2 , NaCl, H 2 S, and H 2 O 2 , Compared with adding only the fluorescent probe in the solution, the fluorescence intensity did not change. When the two Cys and GHS amino acids were added to the solution, the fluorescence of the fluorescent probe increased by 16.5 times after the response to Cys, and the fluorescence increased after the response to GSH. 29.7 times, which shows that the fluorescent probe shows high sensitivity and selectivity for the recognition of cysteine and glutathione. It also shows that the fluorescent probe can be used for cysteine and glutathione in the environment. Acid fluorescence detection and analysis;

[0071] 2. Such as Picture 9 , Picture 10 , Figure 13 with Figure 14 As shown, when the cysteine concentration is in the range of 0-300μM, the fluorescence intensity increases as the concentration of cysteine increases, and in the range of 0-60min, the cysteine that responds to the fluorescent probe As the amount of acid increases, the fluorescence intensity gradually increases;

[0072] 3. Such as Picture 11 , Picture 12 , Figure 15 with Figure 16 As shown, when the glutathione concentration is in the range of 0-300μM, the fluorescence intensity increases with the increase of glutathione concentration, and in the range of 0-60min, the glutathione that responds to the fluorescent probe As the amount of peptide acid increases, the fluorescence intensity gradually increases;

[0073] 4. Such as Figure 17 , 18 It can be shown that in the range of Ph 5 to 11, the fluorescent probe shows a certain fluorescence intensity after responding to cysteine and glutathione. Detection and analysis are suitable for the detection environment under different Ph conditions.

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Description & Claims & Application Information

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Classification and recommendation of technical efficacy words

Mild reaction conditions
The synthetic route is simple

Fluorescent probe for identifying cysteine and glutathione as well as preparation method and application thereof

A technology of glutathione and cysteine, applied in the field of chemical analysis and detection, can solve the problem of not having the function of targeting lysosomes, and achieve the effects of high sensitivity, mild reaction conditions and simple synthesis route

AI-Extracted Technical Summary

Problems solved by technology

Method used

Abstract

Image

Examples

Example Embodiment

Example Embodiment

Example Embodiment

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Description & Claims & Application Information

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