A cysteine-specific CPL response material based on rare earth helicene and a preparation method thereof
By designing a helical structure of rare earth helical material to react with cysteine and form a thiazoline ring, the problem of insufficient selective recognition of cysteine by rare earth europium complexes was solved, achieving highly selective and sensitive cysteine detection and promoting its application in the biomedical field.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- HEILONGJIANG UNIV
- Filing Date
- 2026-04-15
- Publication Date
- 2026-07-10
AI Technical Summary
Existing rare earth europium complexes have limited selective recognition capabilities for cysteine, especially lacking a specific CPL response mechanism in the field of biosensing, resulting in complex and expensive detection methods.
A cysteine-specific CPL-responsive material based on rare-earth helices (structural formula (HNEt3)2[Eu2L4]) was designed to achieve high selectivity and high sensitivity detection by using chiral recognition sites in the helical structure to undergo nucleophilic addition-cyclization reactions with the thiol and amino functional groups of cysteine to form a thiazolidinyl ring.
It achieves highly selective and sensitive detection of cysteine, enabling the detection of trace amounts of cysteine in biological samples, simplifying the operation process, and is suitable for real-time detection in biological fluids, thus promoting the application of chiral optical sensing technology in the biomedical field.
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Figure CN122355840A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a cysteine CPL responsive material and its preparation method. Background Technology
[0002] Amino acids are the basic building blocks of proteins and play a vital role in life activities. Cysteine (Cys), a naturally occurring amino acid containing a sulfhydryl group, not only participates in protein structure maintenance and functional regulation but also plays a crucial role in redox balance, signal transduction, and detoxification. Accurate detection of cysteine concentration in biological systems is of great significance for disease diagnosis, drug metabolism research, and basic life science research.
[0003] Currently, methods for detecting cysteine mainly include high-performance liquid chromatography (HPLC), electrochemical methods, and fluorescence spectroscopy. However, these methods generally suffer from limitations such as complex operation, expensive instruments, or lack of chiral selectivity. Chiral circularly polarized luminescence (CPL) technology, as an emerging optical analysis method, can simultaneously provide information on the optical activity and luminescence of substances, exhibiting unique advantages in the field of chiral molecule recognition. Rare-earth europium complexes have become a research hotspot in the field of CPL sensing due to their unique luminescent properties (such as long fluorescence lifetime, narrow emission peak, and high color purity) and chiral response characteristics. However, existing rare-earth europium complexes have limited selective recognition capabilities for natural amino acids, especially the specific CPL response mechanism for cysteine remains unclear, which restricts their application in the field of biosensing. Summary of the Invention
[0004] To address the lack of specificity of rare earth europium complexes for cysteine detection in existing technologies, this invention provides a cysteine-specific CPL-responsive material based on rare earth helices and its preparation method. This CPL-responsive material achieves highly selective and sensitive detection of cysteine through CPL spectral changes.
[0005] The structural formula of the cysteine-specific CPL-responsive material based on rare-earth spirochetes in this invention is as follows:
[0006] ;
[0007] The general structural formula of the cysteine-specific CPL-responsive material based on rare-earth spirochetes is (HNEt3)2[Eu2L4], where L is a ligand, and the structural formula of L is:
[0008] .
[0009] The preparation method of the cysteine-specific CPL-responsive material based on rare earth spirochetes of the present invention is carried out according to the following steps:
[0010] Step 1: Synthesis of 2,2'-dimethyl-1,1'-biphenyl
[0011] Under nitrogen protection, 5-6 g of 2,2'-dihydroxy-1,1'-biphenyl was dissolved in 60-70 mL of anhydrous N,N-dimethylformamide. 6-8.27 g of anhydrous K2CO3 powder was added to the reaction system in batches. The reaction system was stirred at 50 °C for 1 hour, and then 4-6 mL of CH3I was added. After the reaction was complete, 220-240 mL of water was added to quench the reaction. The resulting reaction solution was mixed with 350 mL of 10% NaOH solution and stirred for 2 hours. The reaction solution was then extracted multiple times with CH2Cl2, washed with water, and the organic phases were combined and dried with Na2SO4. 2,2'-dimethyl-1,1'-biphenyl was obtained by vacuum filtration.
[0012] Step 2: Synthesis of 3,3'-diacetyl-6,6'-dimethylbiphenyl
[0013] 2-3 g of 2,2'-dimethyl-1,1'-biphenyl was dissolved in 5-10 mL of dry 1,2-dichloroethane solution at 0 °C. Then, 1-2 g of acetyl chloride, 2-3 g of anhydrous AlCl3, and 35 mL of 1,2-dichloroethane were mixed and added to the reaction solution, and stirred at 0 °C for 4 hours. The reaction temperature was raised to room temperature and the reaction was continued for 16 hours. After the reaction, the entire reaction system was added to 200-300 mL of ice water. The organic layers were collected and combined and dried with Na2SO4. The inorganic insoluble matter was removed by vacuum filtration. The product was concentrated to 50% of its original volume under reduced pressure to remove 1,2-dichloroethane. Finally, the product was recrystallized from anhydrous ethanol to obtain 3,3'-diacetyl-6,6'-dimethylbiphenyl.
[0014] Step 3: Synthesis of 3,3'-diacetyl-6,6'-dihydroxybiphenyl
[0015] Dissolve 5-6 g of 3,3'-diacetyl-6,6'-dimethylbiphenyl in 100-200 mL of dry 1,2-dichloroethane at room temperature; then add 7-9 g of anhydrous aluminum trichloride and reflux at 90 °C for 25 min; remove inorganic insoluble matter by vacuum filtration, concentrate to 50% of the original volume to remove 1,2-dichloroethane, extract with NaOH solution, wash with 50-100 mL of water, adjust pH to 5-6, filter the resulting white precipitate and dry under vacuum to obtain 3,3'-diacetyl-6,6'-dihydroxybiphenyl;
[0016] Step 4: Synthesis of 3,3'-diacetyl-6,6'-bis(2,2-dimethoxyethoxy)biphenyl
[0017] In a 250 mL Schlenk flask under N2 atmosphere, 100–200 mL of N,N-dimethylformamide was added, followed by 4–6 g of 3,3'-diacetyl-6,6'-dihydroxybiphenyl and 17–19 g of Cs2CO3. The mixture was stirred at 110 °C for 0.5 h, and then 11–13 g of 2-bromo-1,1-dimethoxyethane was added. The reaction mixture was reacted for 5 h. The reaction solution was cooled to room temperature and then poured into 10–20 mL of ice water. The crude product was collected and extracted with 100–200 mL of CH2Cl2. The organic layers were combined, washed with saturated NaCl aqueous solution, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to remove the solvent. Finally, the product was purified by silica gel column chromatography to obtain 3,3'-diacetyl-6,6'-bis(2,2-dimethoxyethoxy)biphenyl.
[0018] In the silica gel column chromatography method, the volume ratio of petroleum ether to ethyl acetate is 3:1.
[0019] Step 5: Synthesis of 3,3'-trifluoro-1,3-dioxobutyl-6,6'-bis(2,2-dimethoxyethoxy)biphenyl:
[0020] Weigh 0.1-0.2 g sodium methoxide and 0.05-0.1 g ethyl heptafluorobutyrate and dissolve them in 10-30 mL dimethyl ether and stir until clear. Add 0.1-0.2 g 3,3'-diacetyl-6,6'-bis(2,2-dimethoxyethoxy)biphenyl and stir at room temperature for 24 hours. After the reaction is complete, pour the reaction solution into water and adjust the pH to 2-3. Let it stand to precipitate a light yellow solid. Filter the precipitated yellow solid and wash it with water. Finally, dry it to obtain 3,3'-trifluoro-1,3-dioxobutyl-6,6'-bis(2,2-dimethoxyethoxy)biphenyl.
[0021] Step Six: Synthesis of Ligand L:
[0022] 0.5–0.6 g of 3,3'-trifluoro-1,3-dioxobutyl-6,6'-bis(2,2-dimethoxyethoxy)biphenyl was dissolved in 10–30 mL of 1,4-dioxane, followed by the addition of 0.50 mL of 6 mol / L HCl. The mixture was stirred at 105 °C for 30 min. After the reaction was complete, the reaction solution was poured into ice water, filtered under reduced pressure to obtain a solid, washed with water, and finally recrystallized to obtain ligand L.
[0023] Step 7: Synthesis of (HNEt3)2[Eu2L4]
[0024] 0.2-0.3 g of ligand L and 0.06-0.07 g of triethylamine were dissolved in 8-10 mL of methanol to obtain a reaction solution. The reaction solution was stirred until it became clear. At room temperature, rare earth trifluoromethanesulfonic acid salt was dissolved in 10-12 mL of methanol to obtain a rare earth salt solution. The rare earth salt solution was added to the clear reaction solution. After the reaction solution became completely clear, the mixture was stirred for 24-30 hours. Then, the reaction solution was added to water to form a flocculent precipitate. After standing for a period of time, the precipitate was filtered to obtain (HNEt3)2[Eu2L4].
[0025] Compared with the prior art, the present invention has the following advantages:
[0026] 1. High Selectivity: The cysteine-specific CPL-responsive material based on rare earth helices of this invention can undergo nucleophilic addition-cyclization reactions with the thiol and amino functional groups of cysteine to form a thiazolidinyl ring due to the chiral recognition sites of its helical structure. Through the induced fit effect, the helical skeleton structure is adjusted and chiral information is efficiently transmitted, achieving specific recognition of the natural amino acid cysteine. Compared with other amino acids (alanine, valine, proline, lysine, histidine, tryptophan, threonine, glutamic acid, phenylalanine, methionine, glycine, arginine, tyrosine, leucine, serine, aspartic acid, isoleucine, serine, asparagine), the CPL response signal of cysteine is significantly different, which can effectively eliminate the interference of other amino acids in the biological system.
[0027] 2. High sensitivity: The CPL signal intensity change of the cysteine-specific CPL-responsive material based on rare earth spirochetes in this invention is highly sensitive to cysteine concentration within the range of 0~1.4×10⁻⁶. -3 It exhibits good linearity in the range of mol / L, with a detection limit as low as 10. - 7 mol / L, meeting the requirements for detecting trace amounts of cysteine in biological samples.
[0028] 3. Simple operation: This invention does not require complicated sample pretreatment. Simply mix the complex solution with the sample to be tested, and it can be directly detected by a CPL spectrometer. The detection process can be completed within 30 minutes.
[0029] 4. Wide range of applications: The cysteine-specific CPL-responsive material based on rare earth spirochetes of this invention can be used for real-time detection of cysteine in biological fluids (such as serum and urine), providing a new analytical tool for disease (such as cystinuria, liver cancer, etc.) and drug metabolism research, and promoting the application of chiral optical sensing technology in the biomedical field. Attached Figure Description
[0030] Figure 1The emission spectra of (HNEt3)2[Eu2L4] in CH3OH / HEPES solution with 40 equivalents of different chiral amino acids are shown.
[0031] Figure 2 The CPL spectra of (HNEt3)2[Eu2L4] in CH3OH / HEPES solution with 40 equivalents of different chiral amino acids are shown.
[0032] Figure 3 The graph shows the luminescence enhancement ratio (|ΔI| / |ΔI0|) of (HNEt3)2[Eu2L4] for different chiral amino acids;
[0033] Figure 4 (HNEt3)2[Eu2L4] containing 40 equivalents of L-cysteine (L-Cys) in CH3OH / HEPES buffer. lum (Light blue column) and the luminescent asymmetry factor (g) after adding 40 equivalents of different chiral amino acids (HNEt3)2[Eu2L4). lum The chart shows the changes in (dark blue bars);
[0034] Figure 5 Add 1.4 × 10⁻⁶ ppm of (HNEt₃)₂[Eu₂L₄] to a CH₃OH / HEPES solution. -3 Time-dependent CPL spectra after ML-cysteine (L-Cys);
[0035] Figure 6 Add 1.4 × 10⁻⁶ ppm of (HNEt₃)₂[Eu₂L₄] to a CH₃OH / HEPES solution. -3 Time curve of CPL signal intensity change (ΔI) at 593 nm after ML-cysteine (L-Cys);
[0036] Figure 7 The CPL response of (HNEt3)2[Eu2L4] to 0-1.6 mM L-cysteine (L-Cys) in CH3OH / HEPES solution after 20 min of reaction is shown in the figure.
[0037] Figure 8 The graph shows the linear fit between the signal intensity change (ΔI) of (HNEt3)2[Eu2L4] in CH3OH / HEPES solution for 0-1.6 mM L-cysteine (L-Cys) at 593 nm after 20 min of reaction and the concentration of L-Cys.
[0038] Figure 9The CPL response of (HNEt3)2[Eu2L4] to 0.02-0.1 mM L-cysteine (L-Cys) in CH3OH / HEPES solution after 20 min of reaction is shown in the figure.
[0039] Figure 10 The graph shows the linear fit between the signal intensity change (ΔI) of (HNEt3)2[Eu2L4] in CH3OH / HEPES solution for 0.02-0.1 mM L-cysteine (L-Cys) and the concentration of L-Cys after 20 min of reaction. Detailed Implementation
[0040] The technical solution of the present invention is not limited to the specific embodiments listed below, but also includes any reasonable combination of the specific embodiments.
[0041] Specific Implementation Method 1: This implementation method
[0042] This embodiment has the following beneficial effects:
[0043] 1. High Selectivity: The cysteine-specific CPL-responsive material based on rare earth helices in this embodiment can undergo nucleophilic addition-cyclization reactions with the thiol and amino functional groups of cysteine to form a thiazolidinyl ring due to the chiral recognition sites of its helical structure. Through the induced fit effect, the helical skeleton structure is adjusted and chiral information is efficiently transmitted, achieving specific recognition of the natural amino acid cysteine. Compared with other amino acids (alanine, valine, proline, lysine, histidine, tryptophan, threonine, glutamic acid, phenylalanine, methionine, glycine, arginine, tyrosine, leucine, serine, aspartic acid, isoleucine, serine, asparagine), the CPL response signal of cysteine is significantly different, which can effectively eliminate the interference of other amino acids in the biological system.
[0044] 2. High sensitivity: The CPL polarization degree change of the cysteine-specific CPL-responsive material based on rare-earth helices in this embodiment is highly sensitive to cysteine concentration within the range of 0~1.4×10⁻⁶. -3 It exhibits good linearity in the range of mol / L, with a detection limit as low as 10. -7 mol / L, meeting the requirements for detecting trace amounts of cysteine in biological samples.
[0045] 3. Simple operation: This method does not require complicated sample pretreatment. Simply mix the complex solution with the sample to be tested, and it can be directly detected by a CPL spectrometer. The detection process can be completed within 30 minutes.
[0046] 4. Wide range of applications: The cysteine-specific CPL-responsive material based on rare earth spirochetes in this embodiment can be used for real-time detection of cysteine in biological fluids (such as serum and urine), providing a new analytical tool for disease (such as cystinuria, liver cancer, etc.) and drug metabolism research, and promoting the application of chiral optical sensing technology in the biomedical field.
[0047] Specific Implementation Method Two: The preparation method of the cysteine-specific CPL-responsive material based on rare earth spirochetes in this implementation method is carried out according to the following steps:
[0048] Step 1: Synthesis of 2,2'-dimethyl-1,1'-biphenyl
[0049] Under nitrogen protection, 5-6 g of 2,2'-dihydroxy-1,1'-biphenyl was dissolved in 60-70 mL of anhydrous N,N-dimethylformamide. 6-8.27 g of anhydrous K2CO3 powder was added to the reaction system in batches. The reaction system was stirred at 50 °C for 1 hour, and then 4-6 mL of CH3I was added. After the reaction was complete, 220-240 mL of water was added to quench the reaction. The resulting reaction solution was mixed with 350 mL of 10% NaOH solution and stirred for 2 hours. The reaction solution was then extracted multiple times with CH2Cl2, washed with water, and the organic phases were combined and dried with Na2SO4. 2,2'-dimethyl-1,1'-biphenyl was obtained by vacuum filtration.
[0050] Step 2: Synthesis of 3,3'-diacetyl-6,6'-dimethylbiphenyl
[0051] 2-3 g of 2,2'-dimethyl-1,1'-biphenyl was dissolved in 5-10 mL of dry 1,2-dichloroethane solution at 0 °C. Then, 1-2 g of acetyl chloride, 2-3 g of anhydrous AlCl3, and 35 mL of 1,2-dichloroethane were mixed and added to the reaction solution, and stirred at 0 °C for 4 hours. The reaction temperature was raised to room temperature and the reaction was continued for 16 hours. After the reaction, the entire reaction system was added to 200-300 mL of ice water. The organic layers were collected and combined and dried with Na2SO4. The inorganic insoluble matter was removed by vacuum filtration. The product was concentrated to 50% of its original volume under reduced pressure to remove 1,2-dichloroethane. Finally, the product was recrystallized from anhydrous ethanol to obtain 3,3'-diacetyl-6,6'-dimethylbiphenyl.
[0052] Step 3: Synthesis of 3,3'-diacetyl-6,6'-dihydroxybiphenyl
[0053] Dissolve 5-6 g of 3,3'-diacetyl-6,6'-dimethylbiphenyl in 100-200 mL of dry 1,2-dichloroethane at room temperature; then add 7-9 g of anhydrous aluminum trichloride and reflux at 90 °C for 25 min; remove inorganic insoluble matter by vacuum filtration, concentrate to 50% of the original volume to remove 1,2-dichloroethane, extract with NaOH solution, wash with 50-100 mL of water, adjust pH to 5-6, filter the resulting white precipitate and dry under vacuum to obtain 3,3'-diacetyl-6,6'-dihydroxybiphenyl;
[0054] Step 4: Synthesis of 3,3'-diacetyl-6,6'-bis(2,2-dimethoxyethoxy)biphenyl
[0055] In a 250 mL Schlenk flask under N2 atmosphere, 100–200 mL of N,N-dimethylformamide was added, followed by 4–6 g of 3,3'-diacetyl-6,6'-dihydroxybiphenyl and 17–19 g of Cs2CO3. The mixture was stirred at 110 °C for 0.5 h, and then 11–13 g of 2-bromo-1,1-dimethoxyethane was added. The reaction mixture was reacted for 5 h. The reaction solution was cooled to room temperature and then poured into 10–20 mL of ice water. The crude product was collected and extracted with 100–200 mL of CH2Cl2. The organic layers were combined, washed with saturated NaCl aqueous solution, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to remove the solvent. Finally, the product was purified by silica gel column chromatography to obtain 3,3'-diacetyl-6,6'-bis(2,2-dimethoxyethoxy)biphenyl.
[0056] In the silica gel column chromatography method, the volume ratio of petroleum ether to ethyl acetate is 3:1.
[0057] Step 5: Synthesis of 3,3'-trifluoro-1,3-dioxobutyl-6,6'-bis(2,2-dimethoxyethoxy)biphenyl:
[0058] Weigh 0.1-0.2 g sodium methoxide and 0.05-0.1 g ethyl heptafluorobutyrate and dissolve them in 10-30 mL dimethyl ether and stir until clear. Add 0.1-0.2 g 3,3'-diacetyl-6,6'-bis(2,2-dimethoxyethoxy)biphenyl and stir at room temperature for 24 hours. After the reaction is complete, pour the reaction solution into water and adjust the pH to 2-3. Let it stand to precipitate a light yellow solid. Filter the precipitated yellow solid and wash it with water. Finally, dry it to obtain 3,3'-trifluoro-1,3-dioxobutyl-6,6'-bis(2,2-dimethoxyethoxy)biphenyl.
[0059] Step Six: Synthesis of Ligand L:
[0060] 0.5–0.6 g of 3,3'-trifluoro-1,3-dioxobutyl-6,6'-bis(2,2-dimethoxyethoxy)biphenyl was dissolved in 10–30 mL of 1,4-dioxane, followed by the addition of 0.50 mL of 6 mol / L HCl. The mixture was stirred at 105 °C for 30 min. After the reaction was complete, the reaction solution was poured into ice water, filtered under reduced pressure to obtain a solid, washed with water, and finally recrystallized to obtain ligand L.
[0061] Step 7: Synthesis of (HNEt3)2[Eu2L4]
[0062] 0.2-0.3 g of ligand L and 0.06-0.07 g of triethylamine were dissolved in 8-10 mL of methanol to obtain a reaction solution. The reaction solution was stirred until it became clear. At room temperature, rare earth trifluoromethanesulfonic acid salt was dissolved in 10-12 mL of methanol to obtain a rare earth salt solution. The rare earth salt solution was added to the clear reaction solution. After the reaction solution became completely clear, the mixture was stirred for 24-30 hours. Then, the reaction solution was added to water to form a flocculent precipitate. After standing for a period of time, the precipitate was filtered to obtain (HNEt3)2[Eu2L4].
[0063] 1. High Selectivity: The cysteine-specific CPL-responsive material based on rare earth helices in this embodiment can undergo nucleophilic addition-cyclization reactions with the thiol and amino functional groups of cysteine to form a thiazolidinyl ring due to the chiral recognition sites of its helical structure. Through the induced fit effect, the helical skeleton structure is adjusted and chiral information is efficiently transmitted, achieving specific recognition of the natural amino acid cysteine. Compared with other amino acids (alanine, valine, proline, lysine, histidine, tryptophan, threonine, glutamic acid, phenylalanine, methionine, glycine, arginine, tyrosine, leucine, serine, aspartic acid, isoleucine, serine, asparagine), the CPL response signal of cysteine is significantly different, which can effectively eliminate the interference of other amino acids in the biological system.
[0064] 2. High sensitivity: The CPL polarization degree change of the cysteine-specific CPL-responsive material based on rare-earth helices in this embodiment is highly sensitive to cysteine concentration within the range of 0~1.4×10⁻⁶. -3 It exhibits good linearity in the range of mol / L, with a detection limit as low as 10. -7 mol / L, meeting the requirements for detecting trace amounts of cysteine in biological samples.
[0065] 3. Simple operation: This method does not require complicated sample pretreatment. Simply mix the complex solution with the sample to be tested, and it can be directly detected by a CPL spectrometer. The detection process can be completed within 30 minutes.
[0066] 4. Wide range of applications: The cysteine-specific CPL-responsive material based on rare earth spirochetes in this embodiment can be used for real-time detection of cysteine in biological fluids (such as serum and urine), providing a new analytical tool for disease (such as cystinuria, liver cancer, etc.) and drug metabolism research, and promoting the application of chiral optical sensing technology in the biomedical field.
[0067] Specific Implementation Method 3: This implementation method differs from Specific Implementation Method 2 in that: after the reaction in step six is completed, the reaction solution is poured into 10~30 mL of ice water.
[0068] Specific Implementation Method Four: This implementation method differs from Specific Implementation Method Two in that: in step seven, the reaction solution is added to 10-20 mL of water to form a flocculent precipitate.
[0069] Specific Implementation Method 5: This implementation method differs from Specific Implementation Method 2 in that: in step 3, 100-200 mL of 5% NaOH solution is used for extraction.
[0070] Specific Implementation Method Six: This implementation method differs from Specific Implementation Method Two in that step three uses 1.0 mol / L hydrochloric acid to adjust the pH.
[0071] Specific Implementation Method Seven: This implementation method differs from Specific Implementation Method Two in that hydrochloric acid is used to adjust the pH in step five.
[0072] Specific Implementation Method 8: This implementation method differs from Specific Implementation Method 2 in that the recrystallization in step 6 uses o-xylene.
[0073] Specific Implementation Method Nine: This implementation method differs from Specific Implementation Method Two in that the rare earth trifluoromethanesulfonic acid salt mentioned in step seven is Eu(OTf)3.
[0074] Specific Implementation Method 10: This implementation method differs from Specific Implementation Method 2 in that the mass fraction of rare earth trifluoromethanesulfonic acid salt in the rare earth salt solution described in step 7 is 60-80%.
[0075] Example 1:
[0076] The preparation method of the cysteine-specific CPL-responsive material based on rare earth spirochetes in this embodiment is carried out according to the following steps:
[0077] Step 1: Synthesis of 2,2'-dimethyl-1,1'-biphenyl
[0078] Under nitrogen protection, 5.1 g of 2,2'-dihydroxy-1,1'-biphenyl was dissolved in 70 mL of anhydrous N,N-dimethylformamide. 8.27 g of anhydrous K₂CO₃ powder was added to the reaction system in batches. The reaction system was stirred at 50 °C for 1 hour, followed by the addition of 4 mL of CH₃I. After the reaction was complete, 230 mL of water was added to quench the reaction. The resulting reaction solution was mixed with 350 mL of 10% NaOH solution and stirred for 2 hours. The reaction solution was then extracted multiple times with CH₂Cl₂, washed with water, and the combined organic phases were dried over Na₂SO₄. The resulting product, 2,2'-dimethyl-1,1'-biphenyl, was obtained by vacuum filtration with a yield of 86.90%.
[0079] Step 2: Synthesis of 3,3'-diacetyl-6,6'-dimethylbiphenyl
[0080] 2 g of 2,2'-dimethyl-1,1'-biphenyl was dissolved in 5–10 mL of dry 1,2-dichloroethane solution at 0 °C. Then, 1.68 g of acetyl chloride, 2.86 g of anhydrous AlCl3, and 35 mL of 1,2-dichloroethane were mixed and added to the reaction solution, and the mixture was stirred at 0 °C for 4 hours. The reaction temperature was then increased to room temperature and the reaction was continued for 16 hours. After the reaction, the entire reaction system was added to 250 mL of ice water. The combined organic layers were collected and dried with Na2SO4. The inorganic insoluble matter was removed by vacuum filtration, and the product was concentrated to 50% of its original volume under reduced pressure to remove 1,2-dichloroethane. Finally, the product was recrystallized from anhydrous ethanol to give 3,3'-diacetyl-6,6'-dimethylbiphenyl; the yield was 89.93%.
[0081] Step 3: Synthesis of 3,3'-diacetyl-6,6'-dihydroxybiphenyl
[0082] 5 g of 3,3'-diacetyl-6,6'-dimethylbiphenyl was dissolved in 120 mL of dry 1,2-dichloroethane at room temperature; then 8.95 g of anhydrous aluminum trichloride was added, and the mixture was refluxed at 90 °C for 25 min; inorganic insoluble matter was removed by vacuum filtration, and the mixture was concentrated to 50% of its original volume to remove 1,2-dichloroethane. The solution was extracted twice with 120 mL of 5% NaOH solution, washed twice with 60 mL of water, and the pH was adjusted to 5-6 with 1.0 mol / L hydrochloric acid. The resulting white precipitate was filtered and dried under vacuum to obtain 3,3'-diacetyl-6,6'-dihydroxybiphenyl; the yield was 86.09%.
[0083] Step 4: Synthesis of 3,3'-diacetyl-6,6'-bis(2,2-dimethoxyethoxy)biphenyl
[0084] Under a nitrogen atmosphere, 150 mL of N,N-dimethylformamide was added to a 250 mL Schlenk flask, followed by 5 g of 3,3'-diacetyl-6,6'-dihydroxybiphenyl and 18.08 g of Cs₂CO₃. The mixture was stirred at 110 °C for 0.5 h, and then 12.43 g of 2-bromo-1,1-dimethoxyethane was added, and the reaction was allowed to proceed for 5 h. The reaction solution was cooled to room temperature and then poured into 10 mL of ice water. The crude product was collected and extracted three times with 150 mL of CH₂Cl₂. The organic layers were combined, washed with saturated NaCl aqueous solution, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to remove the solvent. Finally, the product was purified by silica gel column chromatography to obtain 3,3'-diacetyl-6,6'-bis(2,2-dimethoxyethoxy)biphenyl; the yield was 74.28%.
[0085] In the silica gel column chromatography method, the volume ratio of petroleum ether to ethyl acetate is 3:1.
[0086] Step 5: Synthesis of 3,3'-trifluoro-1,3-dioxobutyl-6,6'-bis(2,2-dimethoxyethoxy)biphenyl:
[0087] 0.19 g of sodium methoxide and 0.064 g of ethyl heptafluorobutyrate were weighed and dissolved in 20 mL of dimethyl ether and stirred until clear. 0.2 g of 3,3'-diacetyl-6,6'-bis(2,2-dimethoxyethoxy)biphenyl was added and stirred at room temperature for 24 hours. After the reaction was complete, the reaction solution was poured into water and the pH was adjusted to 2-3 with hydrochloric acid. A pale yellow solid precipitated out. The precipitated yellow solid was filtered, washed with water, and finally dried to obtain 3,3'-trifluoro-1,3-dioxobutyl-6,6'-bis(2,2-dimethoxyethoxy)biphenyl; yield 78.10%.
[0088] Step Six: Synthesis of Ligand L:
[0089] 0.5 g of 3,3'-trifluoro-1,3-dioxobutyl-6,6'-bis(2,2-dimethoxyethoxy)biphenyl was dissolved in 10 mL of 1,4-dioxane, followed by the addition of 0.50 mL of 6 mol / L HCl. The mixture was stirred at 105 °C for 30 min. After the reaction was complete, the reaction solution was poured into 10 mL of ice water and filtered under reduced pressure to obtain a solid. The solid was washed with water and finally recrystallized from o-xylene to obtain ligand L.
[0090] Step 7: Synthesis of (HNEt3)2[Eu2L4]
[0091] 0.2 g of ligand L and 0.065 g of triethylamine were dissolved in 10 mL of methanol to obtain a reaction solution. The reaction solution was stirred until it became clear. At room temperature, rare earth trifluoromethanesulfonic acid salt was dissolved in 11 mL of methanol to obtain a rare earth salt solution. The rare earth salt solution was added to the clear reaction solution. After the reaction solution became completely clear, the mixture was stirred for 24 hours. Then, the reaction solution was added to 20 mL of water to form a flocculent precipitate. After standing for a period of time, the precipitate was filtered to obtain (HNEt3)2[Eu2L4].
[0092] The rare earth trifluoromethanesulfonic acid salt is Eu(OTf)3;
[0093] The rare earth salt solution contains 65% rare earth trifluoromethanesulfonate by mass.
[0094] The structural formula of the cysteine-specific CPL-responsive material based on rare earth spirochetes prepared in this embodiment is as follows:
[0095] ;
[0096] The general structural formula of the cysteine-specific CPL-responsive material based on rare-earth spirochetes is (HNEt3)2[Eu2L4], where L is a ligand, and the structural formula of L is:
[0097] .
[0098] Figure 1 The emission spectra of (HNEt3)2[Eu2L4] in CH3OH / HEPES solution with 40 equivalents of different chiral amino acids are shown. Figure 1 The helicate is (HNEt3)2[Eu2L4]; Figure 1 This indicates that changes in emission spectra are insufficient to distinguish between different amino acids.
[0099] Figure 2 The CPL spectra of (HNEt3)2[Eu2L4] in CH3OH / HEPES solution with 40 equivalents of different chiral amino acids are shown. Figure 2 This indicates that (HNEt3)2[Eu2L4] can specifically recognize L-cysteine.
[0100] Figure 3 The graph shows the luminescence enhancement ratio (|ΔI| / |ΔI0|) of (HNEt3)2[Eu2L4] for different chiral amino acids; Figure 3 This indicates that (HNEt3)2[Eu2L4] has significant specific recognition of L-cysteine.
[0101] Figure 4(HNEt3)2[Eu2L4] containing 40 equivalents of L-cysteine (L-Cys) in CH3OH / HEPES buffer. lum (Light blue column) and the luminescent asymmetry factor (g) after adding 40 equivalents of different chiral amino acids (HNEt3)2[Eu2L4). lum The chart shows the changes in (dark blue bars); Figure 4 This indicates that (HNEt3)2[Eu2L4] exhibits excellent anti-interference performance as a CPL sensor for detecting L-cysteine.
[0102] Figure 5 Add 1.4 × 10⁻⁶ ppm of (HNEt₃)₂[Eu₂L₄] to a CH₃OH / HEPES solution. -3 Time-dependent CPL spectra after ML-cysteine (L-Cys); Figure 5 This indicates that the recognition of L-cysteine by (HNEt3)2[Eu2L4] is time-dependent, and the interaction between (HNEt3)2[Eu2L4] and L-cysteine reaches equilibrium after 10 min of action.
[0103] Figure 6 Add 1.4 × 10⁻⁶ ppm of (HNEt₃)₂[Eu₂L₄] to a CH₃OH / HEPES solution. -3 Time curve of CPL signal intensity change (ΔI) at 593 nm after ML-cysteine (L-Cys); Figure 6 This indicates that the recognition of L-cysteine by (HNEt3)2[Eu2L4] is time-dependent, and the interaction between (HNEt3)2[Eu2L4] and L-cysteine reaches equilibrium after 10 min of action.
[0104] Figure 7 The CPL response of (HNEt3)2[Eu2L4] to 0-1.6 mM L-cysteine (L-Cys) in CH3OH / HEPES solution after 20 min of reaction is shown in the figure. Figure 7 This indicates that the CPL signal increases with increasing L-Cys concentration and tends to stabilize after 1.4 mM, and (HNEt3)2[Eu2L4] can be used to quantitatively detect L-cysteine.
[0105] Figure 8 The graph shows the linear fit between the signal intensity change (ΔI) of (HNEt3)2[Eu2L4] in CH3OH / HEPES solution for 0-1.6 mM L-cysteine (L-Cys) at 593 nm after 20 min of reaction and the concentration of L-Cys. Figure 8This indicates that ΔI and L-Cys concentration have a good linear relationship in the range of 0-1.6 mM.
[0106] Detection limit test procedure: Dissolve 1.5 mg of the complex in 0.5 mL of CH3OH, then add 4.5 mL of 0.1 mol / L HEPES buffer solution (adjust pH to 7.7) to prepare a concentration of 10. -4 A mother liquor of a complex with a volume ratio of mol / L CH3OH:H2O of 1:9 was prepared. L-cysteine (L-Cys) was prepared into an aqueous solution, and five different concentrations of aqueous solutions (0.02-0.1 mM) were prepared and added to the above mother liquor in sequence. The reaction was carried out at a constant temperature for 20 min. The reaction solution was injected into a quartz cuvette with an optical path of 1 mm. CPL spectral data were collected in the wavelength range of 570-600 nm using a 375 nm laser as the excitation source. Figure 9 The CPL response of (HNEt3)2[Eu2L4] to 0.02-0.1 mM L-cysteine (L-Cys) in CH3OH / HEPES solution after 20 min of reaction is shown in the figure. Figure 10 The graph shows the linear relationship between the change in signal intensity (ΔI) of (HNEt3)2[Eu2L4] at 593 nm and the concentration of L-cysteine (L-Cys) in CH3OH / HEPES solution after 20 min of reaction for 0.02–0.1 mM L-cysteine (L-Cys). The detection limit of this analytical method was calculated to be 8.11 × 10⁻⁶. -7 M (signal-to-noise ratio S / N=3) further highlights the probe's ability to detect trace amounts of L-Cys.
Claims
1. A cysteine-specific CPL-responsive material based on rare earth spirochetes, characterized in that: The structural formula of the cysteine-specific CPL-responsive material based on rare earth spirochetes is as follows: ； The general structural formula of the cysteine-specific CPL-responsive material based on rare-earth spirochetes is (HNEt3)2[Eu2L4], where L is a ligand, and the structural formula of L is: 。 2. The method for preparing cysteine-specific CPL-responsive materials based on rare-earth spirochetes as described in claim 1, characterized in that: The preparation method of cysteine-specific CPL-responsive materials based on rare earth spirochetes is carried out according to the following steps: Step 1: Synthesis of 2,2'-dimethyl-1,1'-biphenyl Under nitrogen protection, 5-6 g of 2,2'-dihydroxy-1,1'-biphenyl was dissolved in 60-70 mL of anhydrous N,N-dimethylformamide. 6-8.27 g of anhydrous K2CO3 powder was added to the reaction system in batches. The reaction system was stirred at 50 °C for 1 hour, and then 4-6 mL of CH3I was added. After the reaction was complete, 220-240 mL of water was added to quench the reaction. The resulting reaction solution was mixed with 350 mL of 10% NaOH solution and stirred for 2 hours. The reaction solution was then extracted multiple times with CH2Cl2, washed with water, and the organic phases were combined and dried with Na2SO4. 2,2'-dimethyl-1,1'-biphenyl was obtained by vacuum filtration. Step 2: Synthesis of 3,3'-diacetyl-6,6'-dimethylbiphenyl 2-3 g of 2,2'-dimethyl-1,1'-biphenyl was dissolved in 5-10 mL of dry 1,2-dichloroethane solution at 0 °C. Then, 1-2 g of acetyl chloride, 2-3 g of anhydrous AlCl3, and 35 mL of 1,2-dichloroethane were mixed and added to the reaction solution, and stirred at 0 °C for 4 hours. The reaction temperature was raised to room temperature and the reaction was continued for 16 hours. After the reaction, the entire reaction system was added to 200-300 mL of ice water. The organic layers were collected and combined and dried with Na2SO4. The inorganic insoluble matter was removed by vacuum filtration. The product was concentrated to 50% of its original volume under reduced pressure to remove 1,2-dichloroethane. Finally, the product was recrystallized from anhydrous ethanol to obtain 3,3'-diacetyl-6,6'-dimethylbiphenyl. Step 3: Synthesis of 3,3'-diacetyl-6,6'-dihydroxybiphenyl Dissolve 5-6 g of 3,3'-diacetyl-6,6'-dimethylbiphenyl in 100-200 mL of dry 1,2-dichloroethane at room temperature; then add 7-9 g of anhydrous aluminum trichloride and reflux at 90 °C for 25 min; remove inorganic insoluble matter by vacuum filtration, concentrate to 50% of the original volume to remove 1,2-dichloroethane, extract with NaOH solution, wash with 50-100 mL of water, adjust pH to 5-6, filter the resulting white precipitate and dry under vacuum to obtain 3,3'-diacetyl-6,6'-dihydroxybiphenyl; Step 4: Synthesis of 3,3'-diacetyl-6,6'-bis(2,2-dimethoxyethoxy)biphenyl In a 250 mL Schlenk flask under N2 atmosphere, 100–200 mL of N,N-dimethylformamide was added, followed by 4–6 g of 3,3'-diacetyl-6,6'-dihydroxybiphenyl and 17–19 g of Cs2CO3. The mixture was stirred at 110 °C for 0.5 h, and then 11–13 g of 2-bromo-1,1-dimethoxyethane was added. The reaction mixture was reacted for 5 h. The reaction solution was cooled to room temperature and then poured into 10–20 mL of ice water. The crude product was collected and extracted with 100–200 mL of CH2Cl2. The organic layers were combined, washed with saturated NaCl aqueous solution, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to remove the solvent. Finally, the product was purified by silica gel column chromatography to obtain 3,3'-diacetyl-6,6'-bis(2,2-dimethoxyethoxy)biphenyl. In the silica gel column chromatography method, the volume ratio of petroleum ether to ethyl acetate is 3:
1. Step 5: Synthesis of 3,3'-trifluoro-1,3-dioxobutyl-6,6'-bis(2,2-dimethoxyethoxy)biphenyl: Weigh 0.1-0.2 g sodium methoxide and 0.05-0.1 g ethyl heptafluorobutyrate and dissolve them in 10-30 mL dimethyl ether and stir until clear. Add 0.1-0.2 g 3,3'-diacetyl-6,6'-bis(2,2-dimethoxyethoxy)biphenyl and stir at room temperature for 24 hours. After the reaction is complete, pour the reaction solution into water and adjust the pH to 2-3. Let it stand to precipitate a light yellow solid. Filter the precipitated yellow solid and wash it with water. Finally, dry it to obtain 3,3'-trifluoro-1,3-dioxobutyl-6,6'-bis(2,2-dimethoxyethoxy)biphenyl. Step Six: Synthesis of Ligand L: 0.5–0.6 g of 3,3'-trifluoro-1,3-dioxobutyl-6,6'-bis(2,2-dimethoxyethoxy)biphenyl was dissolved in 10–30 mL of 1,4-dioxane, followed by the addition of 0.50 mL of 6 mol / L HCl. The mixture was stirred at 105 °C for 30 min. After the reaction was complete, the reaction solution was poured into ice water, filtered under reduced pressure to obtain a solid, washed with water, and finally recrystallized to obtain ligand L. Step 7: Synthesis of (HNEt3)2[Eu2L4] 0.2-0.3 g of ligand L and 0.06-0.07 g of triethylamine were dissolved in 8-10 mL of methanol to obtain a reaction solution. The reaction solution was stirred until it became clear. At room temperature, rare earth trifluoromethanesulfonic acid salt was dissolved in 10-12 mL of methanol to obtain a rare earth salt solution. The rare earth salt solution was added to the clear reaction solution. After the reaction solution became completely clear, the mixture was stirred for 24-30 hours. Then, the reaction solution was added to water to form a flocculent precipitate. After standing for a period of time, the precipitate was filtered to obtain (HNEt3)2[Eu2L4].
3. The method for preparing cysteine-specific CPL-responsive materials based on rare-earth spirochetes according to claim 2, characterized in that: After the reaction in step six is complete, pour the reaction solution into 10-30 mL of ice water.
4. The method for preparing cysteine-specific CPL-responsive materials based on rare-earth spirochetes according to claim 2, characterized in that: Step 7: Add the reaction solution to 10-20 mL of water to form a flocculent precipitate.
5. The method for preparing cysteine-specific CPL-responsive materials based on rare-earth spirochetes according to claim 2, characterized in that: Step 3 involves extraction using 100-200 mL of a 5% NaOH solution.
6. The method for preparing cysteine-specific CPL-responsive materials based on rare-earth spirochetes according to claim 2, characterized in that: Step 3: Adjust the pH with 1.0 mol / L hydrochloric acid.
7. The method for preparing cysteine-specific CPL-responsive materials based on rare-earth spirochetes according to claim 2, characterized in that: The pH adjustment in step five uses hydrochloric acid.
8. The method for preparing cysteine-specific CPL-responsive materials based on rare-earth spirochetes according to claim 2, characterized in that: The recrystallization described in step six uses o-xylene.
9. The method for preparing cysteine-specific CPL-responsive materials based on rare-earth spirochetes according to claim 2, characterized in that: The rare earth trifluoromethanesulfonic acid salt mentioned in step seven is Eu(OTf)3.
10. The method for preparing cysteine-specific CPL-responsive materials based on rare-earth spirochetes according to claim 2, characterized in that: The mass fraction of rare earth trifluoromethanesulfonic acid salt in the rare earth salt solution described in step seven is 60-80%.