A fluorescent probe capable of simultaneously detecting cysteine and tryptophan, and a synthesis method and application thereof

Abstract

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

Description

Technical Field

[0001] This invention belongs to the field of fluorescence detection of cysteine ​​and tryptophan, and relates to a fluorescent probe that can simultaneously detect cysteine ​​and tryptophan, its synthesis method and application. Background Technology

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

[0003] Common biothiols include cysteine ​​(Cys), homocysteine ​​(Hcy), and glutathione (GSH). Cysteine, as an essential thiol-containing amino acid, is involved in various physiological processes such as protein synthesis, detoxification, metabolism, and post-translational modifications. On the other hand, abnormal cysteine ​​levels are highly correlated with a variety of diseases, including slow growth, hair depigmentation, 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, as one of the essential amino acids for the human body, plays a vital physiological role. First, tryptophan is an important raw material for the synthesis of neurotransmitters, playing a crucial role in maintaining the normal function of the nervous system. Second, tryptophan is a precursor to vitamin B3, playing an important role in maintaining the immune system and skin health. Furthermore, tryptophan participates in various biochemical reactions in the human body, contributing significantly to maintaining normal physiological functions. Therefore, accurately detecting the tryptophan content in liquid chromatography is of great significance for understanding human health, preventing and treating diseases.

[0005] Traditional methods for detecting cysteine ​​include high-performance liquid chromatography (HPLC), spectrophotometry, voltammetry, capillary electrophoresis, and mass spectrometry. Tryptophan liquid chromatography detection techniques are mainly divided into two categories: biochemical methods and spectroscopic methods. Biochemical methods detect tryptophan content through enzymatic reactions, while spectroscopic methods calculate its content by detecting the absorption spectrum of tryptophan molecules at specific wavelengths. However, these methods are complex and expensive, easily affected by interference, and their accuracy and stability need improvement. Fluorescence detection not only overcomes these shortcomings but also offers advantages over traditional methods, such as high sensitivity, strong selectivity, convenient observation, and low toxicity to organisms.

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

[0007] Technical problem to be solved: This invention provides a fluorescent probe capable of simultaneously detecting cysteine ​​and tryptophan, its synthesis method and application, which can accurately and rapidly detect cysteine ​​and tryptophan among serine, glutamic acid, tyrosine, tryptophan, glycine, methionine, glutathione, glutamine and cysteine.

[0008] Technical solution: A fluorescent probe capable of simultaneously detecting cysteine ​​and tryptophan, with the structure shown in compound 1:

[0009]

[0010] The preparation method of the above fluorescent probe is as follows: Step 1, 1 equivalent of DPP-Br and 2 equivalents of 2-amino-3-((2-hexyldecyl)oxy)pyridine are added to dry toluene, heated under nitrogen protection and refluxed until fully dissolved. 6 equivalents of TiCl4 are added to the mixed solution, and 20 equivalents of triethylamine are added while continuing reflux and stirring. After reflux and stirring are continued, and the formation of the intermediate imine is monitored by thin-layer chromatography on silica gel plates, 24 equivalents of boron trifluoride diethyl ether are added. The mixture is then heated and refluxed again, cooled to room temperature, and the reaction mixture is poured into water and extracted with CH2Cl2. The organic layer is dried with anhydrous sodium sulfate, filtered, and the solvent is removed by rotary evaporation. The crude product is then subjected to column chromatography. After chromatography separation, compound 3 was obtained; in step 2, compound 3, 0.3 equivalents of 1,1-bis(diphenylphosphine)ferrocene palladium chloride dichloromethane complex, 17 equivalents of KOAc, and 4 equivalents of p-hydroxyphenylboronic acid were added to dry DMF; the mixture was refluxed under anhydrous and oxygen-free conditions and nitrogen protection; after cooling to room temperature, the solvent was removed and the mixture was separated by column chromatography to obtain compound 2; in step 3, compound 2 and 4 equivalents of 2,4-dinitrobenzenesulfonyl chloride were added to dry dichloromethane and stirred at room temperature; the resulting solution was then injected with 3 equivalents of triethylamine in an ice bath and reacted at room temperature. After the reaction was completed, the solvent was removed by distillation under reduced pressure, and the mixture was separated by column chromatography to obtain a green solid, namely compound 1.

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

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

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

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

[0015] The reaction formula is shown below:

[0016]

[0017] Beneficial Effects: This invention utilizes PPAB, which possesses inherent high fluorescence intensity, high quantum yield, and good fluorescence stability, making it beneficial for biomedical applications. Furthermore, the preparation method is simple and convenient, exhibiting good biocompatibility and safety, and low cost. This invention uses 2,4-dinitrobenzenesulfonyl chloride to undergo a substitution reaction with a hydroxyl group to obtain nitro-substituted benzenesulfonyl PPAB. The prepared fluorescent probe exhibits strong near-infrared fluorescence and good water solubility, displaying a strong fluorescence emission peak near 700 nm, demonstrating excellent fluorescence spectral performance. The prepared fluorescent probe shows significant specificity and sensitivity in the detection of cysteine ​​and tryptophan. Attached Figure Description

[0018] Figure 1 The mass spectrometry (HRMS) spectrum of the PPAB fluorescent probe for detecting cysteine ​​and tryptophan synthesized in this invention is shown.

[0019] Figure 2 The 1H NMR spectrum of the PPAB fluorescent probe for detecting cysteine ​​and tryptophan synthesized in this invention is shown.

[0020] Figure 3 The images show the UV and fluorescence spectra of the molecular fluorescent probe of this invention before and after the reaction with cysteine, where 1 represents before the reaction and 2 represents after the reaction.

[0021] Figure 4 The images show the fluorescence spectra of the molecular fluorescent probe of this invention before and after the reaction with tryptophan, where 1 represents before the reaction and 2 represents after the reaction.

[0022] Figure 5 The present invention is 1 μmol·L -1 The UV spectra of the molecular fluorescent probes after the addition of different concentrations of cysteine, from A to Q, are as follows: concentrations of 0, 50, 100, 200, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, and 1000 μmol·L⁻¹. -1 Cysteine, in N,N-dimethylformamide solution;

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

[0024] Figure 7 The present invention is 1 μmol·L -1 Fluorescence spectra of the molecular fluorescent probe before and after reaction with different concentrations of tryptophan, from A to Q, with concentrations of 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, and 160 μmol·L⁻¹. -1 Cysteine, in N,N-dimethylformamide solution. The x-axis represents wavelength, and the y-axis represents fluorescence intensity;

[0025] Figure 8 This invention demonstrates the selectivity of the fluorescent molecular probe for cysteine, specifically 1 μmol·L⁻¹. -1 The molecular probe of this invention is added at 1000 μmol·L⁻¹ -1 Changes in fluorescence emission intensity and UV absorption after adding different substances (serine, glutamic acid, tyrosine, tryptophan, glycine, methionine, glutathione, glutamine, and cysteine). The UV spectrum clearly shows a significant red shift in the absorption peak after adding cysteine ​​compared to other substances. The fluorescence spectrum also shows a significant quenching of fluorescence at 705 nm after adding cysteine ​​compared to other substances.

[0026] Figure 9 This invention demonstrates the selectivity of the fluorescent molecular probe for tryptophan, specifically 1 μmol·L⁻¹. -1 The molecular probe of this invention is prepared by adding 160 μmol·L⁻¹. -1 Changes in fluorescence emission intensity after adding different substances (serine, glutamic acid, tyrosine, tryptophan, glycine, methionine, glutathione, glutamine).

[0027] Figure 10 The changes in fluorescence emission intensity of the fluorescent molecular probe of this invention after the addition of tryptophan and cysteine ​​demonstrate that the fluorescent molecular probe is effective for the detection of both cysteine ​​and tryptophan, and produces two completely different effects. Detailed Implementation

[0028] The following examples illustrate the invention in more detail. It should be noted that the following embodiments should not be construed as limiting the scope of protection of the invention. Any non-essential improvements and adjustments made to the invention by those skilled in the art based on the above-described invention should still fall within the scope of protection of the invention.

[0029] Unless otherwise specified, the experimental methods used in this invention are conventional methods. Unless otherwise specified, all materials and reagents used in the experiments are commercially available. All reagents used in the examples are commercially available analytical grade 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 the mixture was refluxed at 90 °C for 10 h until the sodium was completely dissolved. Bromobenzonitrile (7.33 g, 41 mmol) was then added, followed by diisopropyl succinate (5.0 mL, 24 mmol) over 20 h. After the reaction was complete and cooled to 60 °C, acetic acid (4.6 mL) and methanol (30 mL) were added. The reaction mixture was filtered and washed with a large amount of methanol to obtain the residue, which, after drying, yielded compound DPP-Br (5.0 g, yield 54%). The structural formula of the product is as follows:

[0033]

[0034] 1 H NMR (495MHz, DMSO-d6, 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-decyl alcohol (16.0 mL, 56 mmol), triphenylphosphine (17.6 g, 64 mmol), and imidazole (4.6 g, 68 mmol) were dissolved in dichloromethane (80 mL) and continuously stirred until cooled to 0 °C. Iodine (16.4 g, 64 mmol) was then added to the mixture, and the mixture was stirred at 0 °C for 15 min. The resulting solution was then stirred at room temperature for 24 h. After the reaction was complete, the reaction mixture was poured into an aqueous sodium thiosulfate solution, and the organic phase was extracted. The extract was dried over anhydrous sodium sulfate and the solvent was removed by vacuum distillation. Finally, using n-hexane as eluent, the solution was purified by column chromatography to obtain a colorless liquid, which was compound 2-hexyl-1-iododecane (17.41 g, yield 73.11%). The structural formula of the product is as follows:

[0037]

[0038] 1H NMR (500MHz, CDCl3, 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 under N2 protection. 60 mL of ultradry DMF was added to the flask, and the mixture was stirred at room temperature for 1 h. 2-Hexyl-1-iododecane (14.8 g, 42 mmol) was added to the reaction flask, and the reaction was carried out under light-protected conditions for 24 h. After the reaction was complete, the product was transferred to a round-bottom flask, dissolved in toluene, and the solvent was removed by vacuum distillation. The organic phase was extracted with saturated brine and ethyl acetate solution, and the solvent was removed by drying with anhydrous sodium sulfate and diatomaceous earth. Column chromatography yielded a green oily liquid, which was 2-amino-3-((2-hexyldecyl)oxy)pyridine (2.44 g, yield 20.3%). The structural formula of the product is as follows:

[0041]

[0042] 1 H NMR (500MHz, CDCl3, 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 under N2 protection. Then, 2-amino-3-((2-hexyldecyl)oxy)pyridine (1.1 g, 3.3 mmol) was dissolved in anhydrous toluene (50 mL) and added to the reaction flask. The reaction was carried out at 110 °C for 40 min. TiCl4 solution (0.4 mL, 3.6 mmol) was added, and the mixture was stirred and refluxed for 10 min. Et3N solution (2 mL, 14 mmol) was added, and the reaction was continued for 2 h. BF3·OEt2 solution (2 mL, 16.22 mmol) was then added, and the temperature was adjusted to 123 °C and stirred and refluxed for 20 h. After the reaction was complete, the organic phase was extracted with water and dichloromethane solution, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation. Finally, the crude product was purified by column chromatography to give compound 3 (110 mg, yield 14.25%). The structural formula of the product is as follows:

[0045]

[0046] 1 H NMR (500MHz, CDCl3, 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)Cl2·CH2Cl2 (4.6 mg, 0.0055 mmol), and p-hydroxyphenylboronic acid (11.03 mg, 0.08 mmol) were added to 1.6 mL of dry DMF under N2 protection. The reaction was carried out at 80 °C for 3–4 h. After the reaction was completed, the mixture was cooled to room temperature, transferred to a round-bottom flask, and the solvent was removed by vacuum distillation. The crude product was purified by column chromatography to give compound 2 (9.1 mg, yield 37.90%). The structural formula of the product is as follows:

[0049]

[0050] 1H NMR (500MHz, CDCl3, 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 mixture was stirred at room temperature for 10 min. Then, triethylamine solution (0.025 mL) was injected into the reaction mixture under ice bath conditions, and the mixture was stirred at 25 °C for 10 h. After the reaction was complete, the reaction mixture was transferred to a round-bottom flask, and the solvent was removed by vacuum distillation. The crude product was purified by column chromatography to give compound 1 (25.59 mg, yield 23.01%). The structural formula of the product is as follows:

[0053]

[0054] Mass spectra (HRMS) and nuclear magnetic resonance spectra of the fluorescent molecular probes prepared in Example 1 1 The H-NMR spectra are as follows: Figure 1 and Figure 2 This demonstrates that the fluorescent probe for detecting cysteine ​​and tryptophan of the present invention has been successfully synthesized.

[0055] Example 2

[0056] Detection of cysteine ​​using fluorescent molecular probes

[0057] The fluorescent molecular probe prepared above was dissolved in N,N-dimethylformamide solution to prepare a solution of 1 μmol·L⁻¹. -1 The probe solution. Add 2 mL of the prepared 1 μmol·L⁻¹ solution to a 3 mL cuvette. -1 The probe solution of this invention was prepared, and then different concentrations of cysteine ​​were added and mixed evenly. The changes in its ultraviolet absorption and fluorescence spectra were then tested, and the results are as follows: Figure 5 , 6 As shown. From Figure 5 It can be seen from the addition of 1000 μmol·L -1 After adding cysteine, a significant red shift was observed in its UV absorption; therefore, 1 μmol·L⁻¹ was selected. -1 The fluorescent molecular probes of this invention were added with 1000 μmol·L⁻¹. -1Among different substances, such as serine, glutamic acid, tyrosine, glycine, methionine, glutathione, and glutamine, only cysteine ​​showed a red shift in ultraviolet absorption compared to other substances. Figure 8 As shown in the middle left image. From... Figure 6 It can be seen from the addition of 1000 μmol·L -1 After the addition of cysteine, its fluorescence emission showed a significant quenching; therefore, 1 μmol·L⁻¹ was chosen. -1 The fluorescent molecular probes of this invention were added with 1000 μmol·L⁻¹. -1 Among different substances, such as serine, glutamic acid, tyrosine, glycine, methionine, glutathione, and glutamine, only cysteine ​​showed quenched fluorescence compared to the other substances. Figure 8 As shown in the middle right figure.

[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 a solution of 1 μmol·L⁻¹. -1 The probe solution. Add 2 mL of the prepared 1 μmol·L⁻¹ solution to a 3 mL cuvette. -1 The probe solution of this invention was prepared, and then different concentrations of tryptophan were added and mixed evenly. The changes in its ultraviolet absorption spectrum were then measured, and the results are as follows: Figure 7 As shown in the figure, the fluorescence of the solution significantly increased after the addition of cysteine, with the increase exceeding twofold. Therefore, 1 μmol·L⁻¹ was chosen. -1 The fluorescent molecular probes of this invention were prepared by adding 160 μmol·L⁻¹. -1 Among different substances, such as serine, glutamic acid, tyrosine, glycine, methionine, glutathione, and glutamine, only tryptophan showed the most significant fluorescence enhancement effect compared to other substances. Figure 9 As shown.

[0061] The above description is merely a preferred embodiment of the present invention, and the scope of protection of the present invention is not limited thereto. Any simple changes or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the scope of the technology disclosed in the present invention shall fall within the scope of protection of the present invention.

Claims

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

2. The method for preparing the fluorescent probe according to claim 1, characterized in that, Step 1, 1 equivalent of DPP-Br and 2 equivalents of 2-amino-3-((2-hexyldecyl)oxy)pyridine into dry toluene under nitrogen protection, heated to reflux, after complete dissolution, 6 equivalents of TiCl4was added into the mixture, continued to reflux and stirred, 20 equivalents of triethylamine was added, continued to reflux and stirred, after monitoring the formation of intermediate imine by thin layer chromatography on silica gel plate, 24 equivalents of boron trifluoride etherate was added, then heated to continue reflux, cooled to room temperature, the reaction mixture was poured into water, extracted with CH2Cl2, the organic layer was dried with anhydrous sodium sulfate, filtered, rotary evaporation to remove the solvent, the obtained crude product was separated by column chromatography to obtain compound 3; Step 2, compound 3, 0.3 equivalent of 1,1-bisdiphenylphosphinyl ferrocene palladium dichloromethane complex, 17 equivalents of KOAc, 4 equivalents of p-hydroxybenzoic acid was added into dry DMF; refluxed under anhydrous and anaerobic nitrogen protection; cooled to room temperature, removed the solvent and separated by column chromatography to obtain compound 2; Step 3, compound 2, 4 equivalents of 2,4-dinitrobenzenesulfonyl chloride was added into dry dichloromethane, stirred at room temperature; then the obtained solution was injected into 3 equivalents of triethylamine in an ice bath, reacted at room temperature, after the reaction was completed, the solvent was distilled off under reduced pressure, separated by column chromatography to obtain green solid, which was compound 1.

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

4.

5. The use of the fluorescent probe of claim 1 in the preparation of a simultaneous detection of cysteine and tryptophan detection reagent.

6. A test reagent capable of detecting simultaneously cysteine and tryptophan, characterized in that, The fluorescent probe of claim 1 is contained.

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