Fluorescent probe capable of simultaneously detecting cysteine and tryptophan as well as synthesis method and application of fluorescent probe

Abstract

The invention discloses a fluorescent probe capable of simultaneously detecting cysteine and tryptophan and a synthesis method and application thereof, the structure of the fluorescent probe is shown as a compound 1, and the prepared fluorescent probe shows relatively strong near-infrared fluorescence and good water solubility, has a strong fluorescence emission peak near the wavelength of 700 nm, and shows good fluorescence spectrum performance. The prepared fluorescent probe shows remarkable specificity and sensitivity in detection of cysteine and tryptophan.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescence detection of cysteine ​​and tryptophan, and relates to a fluorescent probe capable of simultaneously detecting cysteine ​​and tryptophan, a synthesis method and application thereof. Background Art

[0002] In recent years, intracellular biothiols have attracted extensive attention due to their biochemical functions such as cell growth, xenobiotic metabolism, gene regulation, and redox homeostasis.

[0003] Common biological thiols include cysteine ​​(Cys), homocysteine ​​(Hcy) and glutathione (GSH). Among them, cysteine, as an essential thiol-containing amino acid, is involved in a variety of physiological processes such as protein synthesis, detoxification, metabolism and post-translational modification. On the other hand, its abnormal level is highly correlated with a variety of diseases (such as slow growth, hair pigmentation loss, edema, lethargy, liver damage, muscle and fat loss, skin lesions, Alzheimer's disease and cardiovascular disease). Therefore, effective and sensitive monitoring of abnormal cysteine ​​levels under physiological conditions is of great significance for the early diagnosis and treatment of certain diseases.

[0004] Tryptophan is one of the essential amino acids for the human body and plays an important physiological role in the human body. First, tryptophan is an important raw material for synthesizing neurotransmitters and plays an important role in maintaining the normal function of the nervous system. Secondly, tryptophan is also a vitamin B 3 Tryptophan is a precursor of tryptophan, which plays an important role in maintaining the human immune system and skin health. In addition, tryptophan is also involved in a variety of biochemical reactions in the human body and plays an important role in maintaining the normal physiological functions of the human body. Therefore, accurate detection of the content of tryptophan in the liquid phase is of great significance for understanding the health status of the human body, preventing diseases and treating diseases.

[0005] Traditional methods for detecting cysteine ​​include high performance liquid chromatography, spectrophotometry, voltammetry, capillary electrophoresis and mass spectrometry. Tryptophan liquid phase detection technology is mainly divided into two categories: biochemical method and spectroscopic method. The biochemical method detects the tryptophan content through enzymatic reaction, and the spectroscopic method calculates its content by detecting the absorption spectrum of tryptophan molecules at a specific wavelength. However, these methods are complicated and expensive to operate, and are easily interfered with. Their accuracy and stability need to be improved. Fluorescence detection can not only overcome the above shortcomings, but also has the advantages of high sensitivity, strong selectivity, convenient observation, and low toxicity to organisms compared to traditional methods.

[0006] The fluorescent probes currently developed for detecting cysteine ​​and tryptophan have poor water solubility and small Stokes shift. Summary of the invention

[0007] Technical problem to be solved: The present invention provides a fluorescent probe capable of simultaneously detecting cysteine ​​and tryptophan, and a synthesis method and application thereof, which can accurately and quickly detect cysteine ​​and tryptophan among serine, glutamate, tyrosine, tryptophan, glycine, methionine, glutathione, glutamine and cysteine.

[0008] Technical solution: A fluorescent probe that can simultaneously detect cysteine ​​and tryptophan, the structure of which is shown in compound 1:

[0009]

[0010] The preparation method of the fluorescent probe comprises the following steps: Step 1, adding 1 equivalent of DPP-Br and 2 equivalents of 2-amino-3-((2-hexyldecyl)oxy)pyridine into dry toluene, heating under reflux under nitrogen protection, and adding 6 equivalents of TiCl to the mixed solution after the mixture is fully dissolved. 4 , continue to reflux and stir, add 20 equivalents of triethylamine; continue to reflux and stir, and after monitoring the formation of the intermediate product imine by thin layer chromatography silica gel plate, add 24 equivalents of boron trifluoride etherate, then increase the temperature and continue to reflux, cool to room temperature, pour the reaction mixture into water, and add CH 2 Cl 2 Extraction; adding anhydrous sodium sulfate to the organic layer to dry, filtering, and removing the solvent by rotary evaporation, and the obtained crude product is separated by column chromatography to obtain compound 3; step 2, adding compound 3, 0.3 equivalents of 1,1-bis(diphenylphosphinoferrocene)palladium chloride dichloromethane complex, 17 equivalents of KOAc, and 4 equivalents of p-hydroxyphenylboronic acid to dry DMF; reflux in anhydrous and oxygen-free, nitrogen protection; cooling to room temperature, removing the solvent and separating by column chromatography to obtain compound 2; step 3, adding compound 2, 4 equivalents of 2,4-dinitrobenzenesulfonyl chloride to dry dichloromethane, stirring at room temperature; then injecting 3 equivalents of triethylamine into the obtained solution in an ice bath, reacting at room temperature, and after the reaction is completed, the solvent is distilled off under reduced pressure, and a green solid is obtained after column chromatography, namely compound 1.

[0011] Preferably, the molar ratio of DPP-Br to 2-amino-3-((2-hexyldecyl)oxy)pyridine in step 1 is 1:5.

[0012] Preferably, in step 3, the molar ratio of compound 2 to 2,4-dinitrobenzenesulfonyl chloride is 1:4.

[0013] The application of the fluorescent probe in the simultaneous detection of cysteine ​​and tryptophan.

[0014] A detection reagent capable of simultaneously detecting cysteine ​​and tryptophan contains the fluorescent probe.

[0015] The reaction formula is as follows:

[0016]

[0017] Beneficial effects: The present invention adopts PPAB, which has a certain fluorescence intensity and high fluorescence intensity, high quantum yield, good fluorescence stability, is beneficial to biomedical applications, and the preparation method is simple and convenient, has good biocompatibility and safety, and is low in cost. The present invention uses 2,4-dinitrobenzenesulfonyl chloride to undergo a substitution reaction with a hydroxyl group to obtain a benzenesulfonyl PPAB with a nitro substitution. The prepared fluorescent probe exhibits strong near-infrared fluorescence and good water solubility, has a strong fluorescence emission peak near a wavelength of 700nm, and exhibits good fluorescence spectral performance. The prepared fluorescent probe exhibits significant specificity and sensitivity in the detection of cysteine ​​and tryptophan. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The HRMS spectrum of the PPAB fluorescent probe synthesized by the present invention for detecting cysteine ​​and tryptophan;

[0019] Figure 2 The nuclear magnetic resonance hydrogen spectrum of the PPAB fluorescent probe synthesized by the present invention for detecting cysteine ​​and tryptophan;

[0020] Figure 3 The UV spectrum and fluorescence spectrum of the molecular fluorescent probe of the present invention before and after the reaction with cysteine, wherein 1-before reaction, 2-after reaction;

[0021] Figure 4 The fluorescence spectra of the molecular fluorescent probe of the present invention before and after the reaction with tryptophan, wherein 1-before the reaction, 2-after the reaction;

[0022] Figure 5 1 μmol·L -1 UV spectra of the molecular fluorescent probe after adding different concentrations of cysteine. From A to Q, the concentrations are 0, 50, 100, 200, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, and 1000 μmol·L -1 of cysteine, the solvent is N,N-dimethylformamide solution;

[0023] Figure 6 1 μmol·L -1 Fluorescence spectra of the molecular fluorescent probe before and after the reaction with different concentrations of cysteine. From A to Q, the concentrations are 0, 50, 100, 200, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, and 1000 μmol·L-1 of cysteine, the solvent is N,N-dimethylformamide solution. The horizontal axis is the wavelength, and the vertical axis is the fluorescence intensity;

[0024] Figure 7 1 μmol·L -1 Fluorescence spectra of the molecular fluorescent probe before and after reaction with tryptophan at different concentrations. From A to Q, the concentrations are 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, and 160 μmol·L -1 of cysteine, the solvent is N,N-dimethylformamide solution. The horizontal axis is the wavelength, and the vertical axis is the fluorescence intensity;

[0025] Figure 8 is the selectivity of the fluorescent molecular probe of the present invention to cysteine. -1 The molecular probe of the present invention was added with 1000 μmol·L -1 Changes in fluorescence emission intensity and UV absorption after adding different substances (serine, glutamic acid, tyrosine, tryptophan, glycine, methionine, glutathione, glutamine, cysteine). It is obvious from the UV graph that the absorption peak shows a significant red shift after adding cysteine ​​compared with other substances. It can be seen from the fluorescence spectrum that the fluorescence at 705nm is significantly quenched after adding cysteine ​​compared with other substances.

[0026] Fig. 9 is the selectivity of the fluorescent molecular probe of the present invention to tryptophan. -1 The molecular probe of the present invention was added with 160 μmol·L -1 The fluorescence emission intensity changes after the addition of different substances (serine, glutamate, tyrosine, tryptophan, glycine, methionine, glutathione, glutamine).

[0027] Fig.10 The changes in the fluorescence emission intensity of the fluorescent molecular probe of the present invention after adding tryptophan and cysteine ​​prove that the fluorescent molecular probe is effective in detecting cysteine ​​and tryptophan respectively, and has two completely different effects. DETAILED DESCRIPTION

[0028] The following examples are given to illustrate the present invention in more detail. It is necessary to point out that the following examples cannot be interpreted as limiting the scope of protection of the invention. Some non-essential improvements and adjustments made to the present invention by technical personnel skilled in the art based on the above invention content should still fall within the scope of protection of the present invention.

[0029] The experimental methods used in the present invention are conventional methods unless otherwise specified. The materials, reagents, etc. used in the experiments can be obtained from commercial sources unless otherwise specified. All the following reagents used in the examples are commercially available analytically pure or chemically pure.

[0030] Example 1

[0031] (1) Preparation of compound DPP-Br

[0032] Sodium (1.8 g, 79 mmol) and a small amount of ferric chloride were added to tert-amyl alcohol (30 mL) and refluxed at 90 ° C for 10 h until the sodium was completely dissolved. Bromobenzonitrile (7.33 g, 41 mmol) was added, followed by diisopropyl succinate (5.0 mL, 24 mmol) within 20 h. After the reaction was completed and cooled to 60 ° C, acetic acid (4.6 mL) and methanol (30 mL) were added thereto. The reaction mixture was filtered and washed with a large amount of methanol to obtain a filter residue, which was dried to obtain the compound DPP-Br (5.0 g, yield 54%). The product structure is as follows:

[0033]

[0034] 1 H NMR (495 MHz, DMSO-d 6 , 298K): δ [ppm] = 11.38 (s, 2H), 8.43 (d, J = 8.4Hz, 4H), 7.80 (d, J = 8.4Hz, 4H).

[0035] (2) Preparation of compound 2-hexyl-1-iododecane

[0036] 2-Hexyl-1-decanol (16.0mL, 56mmol), triphenylphosphine (17.6g, 64mmol) and imidazole (4.6g, 68mmol) were dissolved in dichloromethane (80mL), stirred continuously and cooled to 0°C. Subsequently, iodine (16.4g, 64mmol) was added to the mixed solution, stirred at 0°C for 15min, and then the resulting solution was stirred at room temperature for 24h. After the reaction was completed, the reaction mixture was poured into an aqueous solution of sodium thiosulfate, the organic phase was extracted, and the solvent was removed by reduced pressure distillation after drying over anhydrous sodium sulfate. Finally, n-hexane was used as the eluent and purified by column chromatography to obtain a colorless liquid, namely the compound 2-hexyl-1-iododecane (17.41g, yield 73.11%). The product structure is as follows:

[0037]

[0038] 1 H NMR (500 MHz, CDCl 3, 295K): δ [ppm] = 3.27 (d, J = 4.5Hz, 2H), 1.45-1.00 (m, 25H), 0.93-0.83 (m, 6H).

[0039] (3) Preparation of compound 2-amino-3-((2-hexyldecyl)oxy)pyridine

[0040] 2-Amino-3-hydroxypyridine (3.8 g, 36 mmol) and NaH (1.16 g, 52 mmol) were placed in a two-necked flask. 2 Protection. Pour 60mL of ultra-dry DMF into the flask and stir at room temperature for 1h. Inject 2-hexyl-1-iododecane (14.8g, 42mmol) into the reaction flask and react for 24h under light-proof conditions. After the reaction is completed, transfer the product to a round-bottom flask, dissolve it in toluene, and remove the solvent by vacuum distillation. Extract with saturated brine and ethyl acetate solution to obtain an organic phase, and dry it with anhydrous sodium sulfate and diatomaceous earth to remove the solvent. Column chromatography gives a green oily liquid, which is the compound 2-amino-3-((2-hexyldecyl)oxy)pyridine (2.44g, yield 20.3%). The product structure is as follows:

[0041]

[0042] 1 H NMR (500 MHz, CDCl 3 , 295K): δ [ppm] = 7.62 (d, J = 5.3Hz, 1H), 6.88 (d, J = 8.3Hz, 1H), 6.59 (dd, J 1 = 7.9Hz, J 2=4.9Hz,1H),4.65(br,2H),3.84(d,J=5.3Hz,2H),1.83-1.75(m,1H),1.48-1.17(m,24H),0.92-0.82(m,6H).

[0043] (3) Preparation of Compound 3

[0044] DPP-Br (292 mg, 0.66 mmol) was placed in a two-necked flask and N 2 Then, 2-amino-3-((2-hexyldecyl)oxy)pyridine (1.1 g, 3.3 mmol) was dissolved in anhydrous toluene (50 mL) and injected into the reaction flask, and reacted at 110°C for 40 min. TiCl 4 The solution (0.4 mL, 3.6 mmol) was stirred and refluxed for 10 min, and Et 3 N solution (2 mL, 14 mmol) was reacted for 2 h and then BF 3 ·OEt 2Solution (2mL, 16.22mmol), then the temperature was adjusted to 123℃ and stirred under reflux for 20h. After the reaction was completed, the organic phase was extracted with water and dichloromethane solution and collected, dried over anhydrous sodium sulfate and then vacuum distilled to remove the solvent. Finally, the crude product was purified by column chromatography to obtain compound 3 (110mg, yield 14.25%). The product structure is as follows:

[0045]

[0046] 1 H NMR (500 MHz, CDCl 3 , 295K): δ [ppm] = 8.34 (d, J = 9.5Hz, 4H), 7.87 (d, J = 6.0Hz, 2H), 7.65 (d, J = 8.5Hz, 4H), 7.25 (d, J = 9.5Hz, 2H), 7.05 (dd, J = 6.0Hz, J = 8.0Hz 2H)

[0047] (4) Preparation of Compound 2

[0048] Compound 3 (23.45 mg, 0.02 mmol), potassium acetate (34 mg, 0.35 mmol), Pd(dppf)Cl 2 ·CH 2 Cl 2 (4.6 mg, 0.0055 mmol), p-hydroxyphenylboronic acid (11.03 mg, 0.08 mmol) were added to dry DMF (1.6 mL), N 2 Protection. React at 80°C for 3-4h. After the reaction is completed, cool to room temperature, transfer the reaction mixture to a round-bottom flask, and remove the solvent by vacuum distillation. The obtained crude reaction product is purified by column chromatography to obtain compound 2 (9.1 mg, yield 37.90%). The product structure is as follows:

[0049]

[0050] 1 H NMR (500 MHz, CDCl 3 ,295K): δ8.56(d,J=8.6Hz,4H),7.87(d,J=6.2Hz,2H),7.71(d,J=8.6Hz,4H),7.58(d,J =8.7Hz, 5H), 7.20 (d, J = 6.8Hz, 2H), 6.99 (dd, J = 7.9, 6.3Hz, 2H), 6.91 (d, J = 8.6Hz, 4H).

[0051] (5) Preparation of Compound 1

[0052] Compound 2 (80 mg, 0.067 mmol) was added to dry dichloromethane (2.5 mL), followed by 2,4-dinitrobenzenesulfonyl chloride (71 mg, 0.27 mmol), and the reaction was stirred at room temperature for 10 min. Triethylamine solution (0.025 mL) was then injected into the reaction mixture under ice bath conditions, and the reaction was stirred at 25 ° C for 10 h. After the reaction was completed, the reaction mixture was transferred to a round-bottom flask and the solvent was removed by vacuum distillation. The obtained crude reaction product was purified by column chromatography to obtain compound 1 (25.59 mg, yield 23.01%). The product structure is as follows:

[0053]

[0054] Mass HRMS spectra and nuclear magnetic resonance of the fluorescent molecular probe prepared in Example 1 1 The H-NMR spectra are shown in Figure 1 and Figure 2 , indicating that the fluorescent probe for detecting cysteine ​​and tryptophan of the present invention was successfully synthesized.

[0055] Example 2

[0056] Detection of Cysteine ​​by Fluorescent Molecular Probe

[0057] The fluorescent molecular probe prepared above was dissolved in N,N-dimethylformamide solution to prepare 1 μmol·L -1 Add 2 mL of the prepared 1 μmol·L probe solution into a 3 mL cuvette. -1 The probe solution of the present invention was then added with different concentrations of cysteine ​​and mixed evenly, and the changes in ultraviolet absorption and fluorescence spectrum were tested. The results are as follows: Figure 5 , 6 As shown. Figure 5 It can be seen that when 1000 μmol·L -1 After adding cysteine, its ultraviolet absorption showed an obvious red shift, so 1 μmol·L -1 The fluorescent molecular probe of the present invention was added with 1000 μmol·L -1 Different substances, such as serine, glutamic acid, tyrosine, glycine, methionine, glutathione, and glutamine, only cysteine ​​has a red shift in ultraviolet absorption compared to other substances, such as Figure 8 As shown in the middle left picture. Figure 6 It can be seen that when 1000 μmol·L -1 After adding cysteine, the fluorescence emission was significantly quenched, so 1 μmol·L -1 The fluorescent molecular probe of the present invention was added with 1000 μmol·L -1Different substances, such as serine, glutamic acid, tyrosine, glycine, methionine, glutathione, and glutamine, only the fluorescence of cysteine ​​was quenched compared to other substances, such as Figure 8 As shown in the middle right picture.

[0058] Example 3

[0059] Detection of tryptophan using fluorescent molecular probes

[0060] The fluorescent molecular probe prepared above was dissolved in N,N-dimethylformamide solution to prepare 1 μmol·L -1 Add 2 mL of the prepared 1 μmol·L probe solution into a 3 mL cuvette. -1 The probe solution of the present invention was then added with tryptophan of different concentrations and mixed evenly, and the changes in its ultraviolet absorption spectrum were tested. The results are as follows: Figure 7 As shown in the figure, after adding cysteine, the fluorescence of the solution was significantly enhanced, and the enhancement amplitude was more than 2 times. Therefore, 1 μmol·L -1 The fluorescent molecular probe of the present invention was added with 160 μmol·L -1 Different substances, such as serine, glutamic acid, tyrosine, glycine, methionine, glutathione, and glutamine, only tryptophan has the most obvious fluorescence enhancement effect compared to other substances, such as Fig. 9 shown.

[0061] The above is only a preferred specific implementation manner of the present invention, and the protection scope of the present invention is not limited thereto. Any simple change or equivalent replacement of the technical solution that can be obviously obtained by any technician familiar with the technical field within the technical scope disclosed in the present invention falls within the protection scope of the present invention.

Claims

1. A fluorescent probe capable of simultaneously detecting cysteine ​​and tryptophan, characterized in that: The structure is shown in compound 1:

2. The method for preparing the fluorescent probe according to claim 1, characterized in that: The steps are as follows: step 1, add 1 equivalent of DPP-Br and 2 equivalents of 2-amino-3-((2-hexyldecyl)oxy)pyridine into dry toluene, heat and reflux under nitrogen protection, add 6 equivalents of TiCl4 to the mixed solution after it is fully dissolved, continue to reflux and stir, add 20 equivalents of triethylamine; continue to reflux and stir, add 24 equivalents of boron trifluoride ether after the formation of the intermediate product imine is monitored by thin layer chromatography silica gel plate, then increase the temperature and continue to reflux, cool to room temperature, pour the reaction mixture into water, and extract with CH2Cl2; add anhydrous sodium sulfate to dry the organic layer, filter, and remove the solvent by rotary evaporation, and the obtained crude product is separated by column chromatography to obtain To compound 3; step 2, compound 3, 0.3 equivalents of 1,1-bis(diphenylphosphinoferrocene)palladium chloride dichloromethane complex, 17 equivalents of KOAc, and 4 equivalents of p-hydroxyphenylboronic acid are added to dry DMF; reflux in anhydrous and oxygen-free, nitrogen protection; cool to room temperature, remove the solvent and separate by column chromatography to obtain compound 2; step 3, compound 2, 4 equivalents of 2,4-dinitrobenzenesulfonyl chloride are added to dry dichloromethane and stirred at room temperature; then the resulting solution is injected with 3 equivalents of triethylamine in an ice bath, reacted at room temperature, and after the reaction is completed, the solvent is distilled off under reduced pressure, and a green solid, namely compound 1, is obtained after column chromatography separation.

3. The method for preparing the fluorescent probe according to claim 2, characterized in that: The molar ratio of DPP-Br to 2-amino-3-((2-hexyldecyl)oxy)pyridine in step 1 is 1:

5.

4. The method for preparing the fluorescent probe according to claim 2, characterized in that: The molar ratio of compound 2 to 2,4-dinitrobenzenesulfonyl chloride in step 3 is 1:

4.

5. Use of the fluorescent probe according to claim 1 in the simultaneous detection of cysteine ​​and tryptophan.

6. A detection reagent capable of simultaneously detecting cysteine ​​and tryptophan, characterized in that: Contains the fluorescent probe according to claim 1.

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