Chiral amino acid CPL probe based on aldehyde group functionalized luminescent rare earth helicoid complex as well as preparation method and application of chiral amino acid CPL probe

Abstract

The invention discloses a chiral amino acid CPL probe based on an aldehyde group functionalized luminescent rare earth spirochete complex as well as a preparation method and application thereof, and belongs to the technical field of biosensing materials. The invention aims to solve the problem that the existing probe has poor selectivity in recognition of sulfydryl amino acid. The chiral amino acid CPL probe is an aldehyde group functionalized water-soluble lanthanide helical complex, side chain aldehyde groups can enhance the water solubility of the helical complex by forming hemiacetal and can also be used as specific recognition sites, the aldehyde groups react with sulfydryl-containing amino acids such as D-penicillamine and L-cysteine to form thiazolidine rings, and the chiral amino acid CPL probe can be used as a specific recognition site. Furthermore, the chirality of D-PA and L-Cys is triggered to be transmitted to a metal center, and the chirality is amplified into a detectable CPL signal. Thiazolidine rings formed by condensation of different amino acids have steric hindrance differences, and the steric hindrance differences can influence spiral chirality, so that the probe still shows excellent selectivity even in a complex system in which a plurality of interfering substances coexist.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biosensing materials, and particularly relates to a chiral amino acid CPL probe and a preparation method and application thereof. BACKGROUND

[0002] Helical structures are the basic characteristics of many biological macromolecules, including DNA double helix, α-helix in proteins, and triple helix structure of collagen. Inspired by nature, chemists have been working on synthesizing artificial helical structures to mimic biological macromolecules for decades, in order to explore and utilize their unique and intelligent response characteristics. Among various compounds, chiral lanthanide helical complexes have attracted much attention due to their unique physical and chemical properties. In particular, the helical chirality of such complexes can produce circularly polarized luminescence (CPL) phenomenon of lanthanide ions. More valuable is that helical complexes with stimulus-responsive chiral induction properties provide a broad prospect for the development of probes based on CPL technology. However, lanthanide helical complexes generally have poor water solubility, which greatly limits their application in the detection of biological molecules such as amino acids and sugars.

[0003] A common strategy to improve the water solubility of coordination supramolecules is to introduce water-soluble groups such as hydroxyl, polyether or sulfonic acid groups on the ligand. In aqueous environment, hydrophobic interaction can promote the selective inclusion of drug molecules, natural products and hydrocarbon compounds in the internal cavity of such assemblies. The resulting host-guest interaction can cause changes in the optical properties of the host or host-guest complex, which can be used for the detection of target substrates. However, for the detection of water-soluble compounds, there is still a big challenge: that is, both the probe and the substrate have high hydration energy in aqueous solution, which will weaken the supramolecular interaction (especially hydrogen bonding) between the host and the guest, and thus significantly reduce the recognition ability and selectivity for the substrate. To solve this problem, dynamic covalent bonds (such as dynamic formation of C=N bond) with reactivity in aqueous solution can be used to enhance the recognition efficiency of the host-guest system, but it is still difficult to achieve high selective detection due to the similar structure and reactivity of various amino acids.

[0004] Thiol amino acids (such as L-cysteine L-Cys, D-penicillamine D-PA) are key active molecules in the body, L-Cys participates in antioxidant stress and signal transduction, and its abnormal concentration is associated with various diseases; D-PA is a chiral drug for the clinical treatment of diseases such as hepatolenticular degeneration, and the chirality purity directly affects the drug efficacy, so thiol amino acid detection and chirality recognition are crucial. The existing detection technologies (spectroscopy, chromatography, electrochemistry, etc.) have obvious limitations: 1. Poor selectivity, easily interfered by reducing substances or other amino acids in biological samples; 2. Difficult chirality recognition, requiring expensive chiral stationary phases or complex derivatization, time-consuming operation; 3. Poor water solubility of probes, which cannot adapt to physiological environments or drug aqueous solution systems; 4. Insufficient sensitivity and stability, which are difficult to meet the needs of trace detection and drug quality control. Therefore, it is urgent to develop a new thiol amino acid detection technology with high selectivity, chirality discrimination ability, good water solubility and precise quantitative performance, in order to be applied to biomedical and drug analysis scenes. SUMMARY

[0005] In order to solve the problem of poor selectivity of existing probes in recognition of thiol amino acids, the application provides a chiral amino acid CPL probe based on aldehyde functionalized luminescent rare earth helicate complex as well as a preparation method and application thereof. The chiral amino acid CPL probe is an aldehyde functionalized water-soluble lanthanide helical complex, the side chain aldehyde group can not only enhance the water solubility of the helical complex by forming a hemiacetal, but also serve as a specific recognition site. These aldehyde groups react with thiol-containing amino acids such as D-penicillamine (D-PA) and L-cysteine (L-Cys) to form thiazolidine rings, which further trigger the chirality transmission to the metal center and amplify the chirality into a detectable CPL signal. Due to the difference in steric hindrance of the thiazolidine rings formed by different amino acids, and the difference will affect the helical chirality, so even in a complex system coexisting with multiple interferents, the probe still shows excellent selectivity. The aldehyde modification strategy provides a new way for detecting amino-containing water-soluble molecules.

[0006] The structural formula of the chiral amino acid CPL probe based on aldehyde functionalized luminescent rare earth helicate complex is as follows:

[0007] ;

[0008] The structural general formula of the rare earth helicate is (HNEt3)2[Eu2L4], and the structural formula of the ligand L is as follows:

[0009] .

[0010] The application is based on the preparation method of the chiral amino acid CPL probe of aldehyde group functionalized luminescent rare earth helicate complex, which is carried out according to the following steps:

[0011] Step one, synthesis of 4-hydroxy-3-iodoacetophenone

[0012] 3~5 g 4-hydroxyacetophenone, 4~6 g iodine and 10~20 mL hydrogen peroxide aqueous solution are added into 100~200 mL water, stirred at room temperature for 24 h, filtered to obtain a solid, and then recrystallized by ethanol to obtain 4-hydroxy-3-iodoacetophenone;

[0013] Step two, synthesis of 1-(3-iodo-4-(2,2-dimethoxyethoxy) phenyl) ethanone

[0014] 4~6 g 4-hydroxy-3-iodoacetophenone and 8~10 g Cs2CO3 are added to a Schlenk flask under N2 atmosphere, then 100~200 mL N,N-dimethylacetamide is added, stirred at 110 ℃ for 0.5 h; then 5~7 g 2-bromo-1,1-dimethoxyethane is added and reacted for 5 h; the product is poured into water for quenching and product extraction; the obtained organic layer is washed with saturated sodium chloride solution; after the organic layer is dried, the solvent is removed by concentration; finally, purification is performed by silica gel column chromatography to obtain 1-(3-iodo-4-(2,2-dimethoxyethoxy) phenyl) ethanone;

[0015] Step three, synthesis of 5-acetyl-2-(2,2-dimethoxyethoxy)-4'-acetylbiphenyl

[0016] 1~3 g 1-(3-iodo-4-(2,2-dimethoxyethoxy) phenyl) ethanone, 0.2~0.4 g Pd(PPh3)4 and 1~2 g 4-acetylphenylboronic acid are placed in a Schlenk flask under N2 atmosphere; 40~60 mL tetrahydrofuran, 20~30 mL aqueous sodium carbonate solution are added in sequence and stirred under reflux, and after 12 h, the reaction liquid is poured into water and extracted with ethyl acetate; the organic phase is combined and washed with saturated aqueous sodium chloride solution, and the organic phase is dried with anhydrous sodium sulfate, and then the solvent is removed by concentration; finally, purification is performed to obtain 5-acetyl-2-(2,2-dimethoxyethoxy)-4'-acetylbiphenyl;

[0017] Step four, synthesis of 5-(3-oxo-4,4,4-trifluorobutenyl)-2-(2,2-dimethoxyethoxy)-4'-(3-oxo-4,4,4-trifluorobutenyl) biphenyl

[0018] Dissolve 1-3 g of sodium methoxide and 3-5 g of ethyl trifluoroacetate in 10-30 mL of dimethyl ether, respectively; finally, add 1-3 g of 5-acetyl-2-(2,2-dimethoxyethoxy)-4'-acetyl biphenyl, stir at room temperature for 24 h; pour the reaction liquid into water, adjust the pH to 2-3 with HCl, and precipitate the solid, then filter under reduced pressure and dry to obtain 5-(3-oxo-4,4,4-trifluorobutenyl)-2-(2,2-dimethoxyethoxy)-4'-(3-oxo-4,4,4-trifluorobutenyl) biphenyl;

[0019] Step five, synthesis of ligand L:

[0020] Dissolve 0.1-0.5 g of 5-(3-oxo-4,4,4-trifluorobutenyl)-2-(2,2-dimethoxyethoxy)-4'-(3-oxo-4,4,4-trifluorobutenyl) biphenyl in 10-30 mL of 1,4-dioxane, add 1-3 mL of hydrochloric acid and reflux for 40 min; after the reaction is completed, pour into 10 mL of ice water, filter under reduced pressure to obtain a solid, wash with water until the pH is 7, and finally recrystallize to obtain ligand L;

[0021] Step six, synthesis of (HNEt3)2[Eu2L4]

[0022] Dissolve 0.3-0.6 g of ligand L and 0.2-0.4 g of triethylamine in 10-20 mL of methanol to obtain a reaction liquid, and stir until the reaction liquid is clear; dissolve the rare earth triflate in 3-5 mL of methanol to obtain a rare earth salt solution under room temperature conditions; add the rare earth salt solution to the reaction liquid, continue to stir for 12-24 hours after the reaction liquid is completely clear, and then add the obtained reaction liquid to 10-20 mL of water to form a flocculent precipitate, filter after standing, and obtain (HNEt3)2[Eu2L4].

[0023] The present application is based on the chiral amino acid CPL probe of aldehyde group functionalized luminescent rare earth helical complex for detecting sulfhydryl amino acid.

[0024] Compared with the prior art, the present application has the following advantages:

[0025] 1. Excellent water solubility and physiological adaptability: the existing lanthanide helical complex generally has poor water solubility, which limits its application in biomolecular detection. In the present application, the aldehyde group forms a hemiacetal structure, so that the probe has good water solubility in the concentration range of 3×10 -5 ~2.5×10 -4 M, and has strong stability (no significant change in luminescence intensity and spectral profile) in a physiological buffer system with pH 6.8-8.2, and can be directly applied to detection scenarios in aqueous solution and physiological environment.

[0026] 2. High selectivity of recognition: Existing amino acid detection probes often face the problem of insufficient selectivity due to the similarity of amino acid structures, making it difficult to distinguish between thiol-containing and non-thiol-containing amino acids and corresponding enantiomers. The present application forms a thiazolidine ring through the specific reaction mechanism of aldehyde group and thiol-containing amino acid, only D-penicillamine (D-PA) and L-cysteine (L-Cys) produce CPL response, and there is no obvious signal for other 6 common amino acids; and D-PA induces negative signal (g lum =-0.23), and L-Cys induces positive signal (g lum =+0.04), achieving accurate differentiation of enantiomers.

[0027] 3. Sensitive CPL signal and excellent quantitative performance: Some existing CPL probes have the problems of weak signal, narrow quantitative range or high detection limit. The CPL signal response of the present application to D-PA is fast, reaching stability in 30 minutes, showing a good linear relationship (R -3 =0.99174) in the concentration range of 0~0.8×10 2 M, and the detection limit is as low as 1.08×10 -5 M, which is much better than existing similar detection methods, and can meet the accurate quantitative demand of trace thiol-containing amino acids.

[0028] 4. Strong anti-interference ability and high practical application value: Existing probes are easily interfered by other biological molecules in complex matrix, and are difficult to be directly applied to actual sample detection. The detection signal of the present application to D-PA can still maintain high intensity in the presence of other 7 kinds of amino acids with equivalent concentration, and the anti-interference performance is outstanding; at the same time, it is successfully applied to the determination of D-PA content in commercial penicillamine tablets, with recovery rate of 96.67%~100.01% and relative standard deviation (RSD) ≤2.3%, without complex sample pretreatment, and has strong practicability.

[0029] 5. General functionalization strategy and strong expandability: The functionalization modification of existing water-soluble probes mostly focuses on improving solubility, and lacks the cooperative design of specific recognition sites. The aldehyde functionalization strategy proposed in the present application not only solves the water solubility problem of lanthanide helix complexes through hemiacetal structure, but also realizes targeted recognition by taking aldehyde group as specific reaction site, providing a general paradigm for designing water-soluble coordination supramolecular probes for amine-containing biological molecules, and expanding the application scenarios of lanthanide helix complexes in the field of chiral biosensing. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 is the emission spectrum of the complex in CH3OH / HEPES solution in the presence of 40 equivalents of different chiral amino acids;

[0031] Figure 2 CPL spectra of the complex in CH3OH / HEPES solution in the presence of 40 equivalents of different chiral amino acids;

[0032] Figure 3 The luminescence enhancement ratio (|ΔI| / |ΔI0|) of the complex to different chiral amino acids;

[0033] Figure 4 The g of the complex containing 40 equivalents of D-penicillamine (D-PA) in CH3OH / HEPES buffer lum The change (green column) of the complex g lum after adding 40 equivalents of different chiral amino acids;

[0034] Figure 5 The time-dependent CPL spectra of the complex in CH3OH / HEPES solution after adding 0.8×10 -3 M D-penicillamine (D-PA);

[0035] Figure 6 The time curve of the CPL signal intensity change (ΔI) at 588 nm of the complex in CH3OH / HEPES solution after adding 0.8×10 -3 M D-penicillamine (D-PA);

[0036] Figure 7 The CPL response of the complex in CH3OH / HEPES solution to 0-1.2 mM D-penicillamine (D-PA) after 30 min of reaction;

[0037] Figure 8 The linear fitting relationship between the signal intensity change value (ΔI) at 588 nm of the complex in CH3OH / HEPES solution after 30 min of reaction and the concentration of D-penicillamine (D-PA) to 0-1.2 mM D-penicillamine (D-PA). DETAILED DESCRIPTION

[0038] The technical solutions of the present application are not limited to the following specific embodiments, but also include any reasonable combination of the specific embodiments.

[0039] Specific embodiment one: The structure of the chiral amino acid CPL probe based on aldehyde group functionalized luminescent rare earth helicate complex in this embodiment is:

[0040] ;

[0041] The structure of the rare earth helicate is (HNEt3)2[Eu2L4], and the structure of the ligand L is:

[0042] .

[0043] The present embodiment has the following advantages:

[0044] 1. Excellent water solubility and physiological adaptability: Existing lanthanide helix complexes generally have poor water solubility, which limits their application in biomolecular detection. The present embodiment forms a hemiacetal structure through an aldehyde group, so that the probe has good water solubility in the range of 3×10 -5 ~2.5×10 -4 M concentration, and has strong stability (no significant change in luminescence intensity and spectral profile) in a physiological buffer system with pH 6.8~8.2, which can be directly applied to detection scenarios in aqueous solution and physiological environment.

[0045] 2. High recognition selectivity: Existing amino acid detection probes often face the problem of insufficient selectivity due to the similarity of amino acid structures, making it difficult to distinguish between thiol-containing and non-thiol-containing amino acids and corresponding enantiomers. The present embodiment forms a thiazolidine ring through the specific reaction mechanism of the aldehyde group and thiol-containing amino acids, only produces a CPL response to D-penicillamine (D-PA) and L-cysteine (L-Cys), and has no obvious signal to the other six common amino acids; and D-PA induces a negative signal (g lum =-0.23), and L-Cys induces a positive signal (g lum =+0.04), achieving precise differentiation of enantiomers.

[0046] 3. Sensitive CPL signal and excellent quantitative performance: Some existing CPL probes have weak signals, narrow quantitative range, or high detection limit. The present embodiment has a rapid CPL signal response to D-PA, reaching stability in 30 minutes, showing a good linear relationship (R -3 =0.99174) in the range of 0~0.8×10 2 M concentration, with a detection limit as low as 1.08×10 -5 M, which is much better than existing similar detection methods, and can meet the precise quantitative needs of trace thiol-containing amino acids.

[0047] 4. Strong anti-interference ability and high practical application value: Existing probes are easily interfered by other biomolecules in complex matrices, and are difficult to be directly applied to actual sample detection. The present embodiment can maintain a very high intensity of detection signal for D-PA in the presence of other seven amino acids at equivalent concentrations, with outstanding anti-interference performance; at the same time, it is successfully applied to the determination of D-PA content in commercial penicillamine tablets, with a recovery rate of 96.67%~100.01% and a relative standard deviation (RSD) ≤2.3%, without complex sample pretreatment, and has strong practicality.

[0048] 5. The functionalization strategy is general and expandable: The existing functionalization modification of water-soluble probes mainly focuses on improving solubility, and lacks the cooperative design of specific recognition sites. The aldehyde functionalization strategy proposed in this embodiment not only solves the water solubility problem of lanthanide helix complexes through hemiacetal structure, but also realizes targeted recognition with aldehyde group as specific reaction site, providing a general paradigm for designing water-soluble biomolecular coordination supramolecular probes facing amine-containing molecules, and expanding the application scenarios of lanthanide helix complexes in the field of chiral biosensing.

[0049] Specific embodiment two: the preparation method of the chiral amino acid CPL probe of the aldehyde functionalized luminescent rare earth helical complex is carried out according to the following steps:

[0050] Step one, synthesis of 4-hydroxy-3-iodoacetophenone

[0051] 3-5 g of 4-hydroxyacetophenone, 4-6 g of elemental iodine and 10-20 mL of hydrogen peroxide aqueous solution were added to 100-200 mL of water, stirred at room temperature for 24 h, filtered to obtain a solid, and then recrystallized by ethanol to obtain 4-hydroxy-3-iodoacetophenone;

[0052] Step two, synthesis of 1-(3-iodo-4-(2,2-dimethoxyethoxy) phenyl) ethanone

[0053] Under N2 atmosphere, 4-6 g of 4-hydroxy-3-iodoacetophenone and 8-10 g of Cs2CO3 were added to a Schlenk flask, then 100-200 mL of N,N-dimethylacetamide was added, stirred at 110°C for 0.5 h; then 5-7 g of 2-bromo-1,1-dimethoxyethane was added and reacted for 5 h; the product was poured into water to quench and the product was extracted; the obtained organic layer was washed with saturated sodium chloride solution; the organic layer was dried and concentrated to remove the solvent; finally, purification was carried out by silica gel column chromatography to obtain 1-(3-iodo-4-(2,2-dimethoxyethoxy) phenyl) ethanone;

[0054] Step three, synthesis of 5-acetyl-2-(2,2-dimethoxyethoxy)-4'-acetylbiphenyl

[0055] In a Schlenk flask, 1-3 g of 1-(3-iodo-4-(2,2-dimethoxyethoxy)phenyl)ethanone, 0.2-0.4 g of Pd(PPh3)4, and 1-2 g of 4-acetylphenylboronic acid were placed under N2atmosphere; 40-60 mL of tetrahydrofuran, 20-30 mL of an aqueous sodium carbonate solution were added successively, and then reflux stirring was performed, after 12 h, the reaction solution was poured into water and extracted with ethyl acetate; the organic phases were combined and washed with saturated aqueous sodium chloride solution, the organic phase was dried using anhydrous sodium sulfate, and then the solvent was removed by concentration; finally, purification was performed to obtain 5-acetyl-2-(2,2-dimethoxyethoxy)-4'-acetyl biphenyl;

[0056] Step four, synthesis of 5-(3-oxo-4,4,4-trifluorobutenyl)-2-(2,2-dimethoxyethoxy)-4'-(3-oxo-4,4,4-trifluorobutenyl)biphenyl

[0057] In 10-30 mL of dimethyl ether, 1-3 g of sodium methoxide and 3-5 g of ethyl trifluoroacetate were dissolved, respectively; finally, 1-3 g of 5-acetyl-2-(2,2-dimethoxyethoxy)-4'-acetyl biphenyl was added, and stirring was performed at room temperature for 24 h; the reaction solution was poured into water, the pH was adjusted to 2-3 with HCl, and the solid was precipitated and then filtered under reduced pressure and dried to obtain 5-(3-oxo-4,4,4-trifluorobutenyl)-2-(2,2-dimethoxyethoxy)-4'-(3-oxo-4,4,4-trifluorobutenyl)biphenyl;

[0058] Step five, synthesis of ligand L:

[0059] 0.1-0.5 g of 5-(3-oxo-4,4,4-trifluorobutenyl)-2-(2,2-dimethoxyethoxy)-4'-(3-oxo-4,4,4-trifluorobutenyl)biphenyl was dissolved in 10-30 mL of 1,4-dioxane, 1-3 mL of hydrochloric acid was added, and reflux was performed for 40 min; after the reaction was completed, 10 mL of ice water was poured, and the solid was filtered under reduced pressure, washed with water until the pH was 7, and finally recrystallized to obtain ligand L;

[0060] Step six, synthesis of (HNEt3)2[Eu2L4]

[0061] Dissolve 0.3-0.6 g of ligand L and 0.2-0.4 g of triethylamine in 10-20 mL of methanol to obtain a reaction solution, and stir until the reaction solution is clear; dissolve the rare earth triflate salt in 3-5 mL of methanol to obtain a rare earth salt solution at room temperature; add the rare earth salt solution to the reaction solution, continue to stir for 12-24 hours after the reaction solution is completely clear, then add the obtained reaction solution to 10-20 mL of water to form a flocculent precipitate, and filter after standing to obtain (HNEt3)2[Eu2L4].

[0062] 1. Excellent water solubility and physiological adaptability: The existing lanthanide helix complexes generally have poor water solubility, which limits their application in biomolecular detection. In the present embodiment, the aldehyde group forms a hemiacetal structure, so that the probe has good water solubility in the concentration range of 3×10 -5 ~2.5×10 -4 M, and has strong stability (no significant change in luminescence intensity and spectral profile) in a physiological buffer system with pH 6.8-8.2, and can be directly applied to detection scenarios in aqueous solution and physiological environment.

[0063] 2. High recognition selectivity: Existing amino acid detection probes often face the problem of insufficient selectivity due to the similarity of amino acid structures, and it is difficult to distinguish between thiol-containing and non-thiol-containing amino acids and corresponding enantiomers. In the present embodiment, the aldehyde group and thiol-containing amino acid specifically form a thiazolidine ring through a reaction mechanism, only D-penicillamine (D-PA) and L-cysteine (L-Cys) produce CPL response, and there is no obvious signal for the other six common amino acids; and D-PA induces a negative signal (g lum =-0.23), and L-Cys induces a positive signal (g lum =+0.04), achieving precise differentiation of enantiomers.

[0064] 3. CPL signal is sensitive and has excellent quantitative performance: Some existing CPL probes have weak signal, narrow quantitative range or high detection limit. The CPL signal response of the present embodiment to D-PA is fast, reaching stability in 30 minutes, showing good linear relationship (R -3 =0.99174) in the concentration range of 0-0.8×10 2 M, and the detection limit is as low as 1.08×10 -5 M, which is much better than existing similar detection methods, and can meet the precise quantitative demand of trace thiol-containing amino acids.

[0065] 4. Strong anti-interference ability and high practical value: The existing probes are easily interfered by other biological molecules in complex matrix, and are difficult to be directly applied to actual sample detection. The detection signal of D-PA can still maintain high intensity in the presence of other 7 kinds of amino acids at equivalent concentration, and the anti-interference performance is outstanding; at the same time, it is successfully applied to the determination of D-PA content in commercial penicillamine tablets, with recovery rate of 96.67%~100.01%, relative standard deviation (RSD) ≤2.3%, without complex sample pretreatment, and strong practicability.

[0066] 5. General and strong expandability of functionalization strategy: The functionalization modification of existing water-soluble probes mainly focuses on improving solubility, and lacks the cooperative design of specific recognition sites. The aldehyde functionalization strategy proposed in this embodiment not only solves the water-solubility problem of lanthanide helix complexes through hemiacetal structure, but also realizes targeted recognition by taking aldehyde group as specific reaction site, which provides a general paradigm for designing coordination supramolecular probes for amine-containing water-soluble biological molecules, and expands the application scenarios of lanthanide helix complexes in the field of chiral biological sensing.

[0067] Specific embodiment three: The difference between this embodiment and specific embodiment two is that the mass fraction of the hydrogen peroxide aqueous solution in step one is 30%.

[0068] Specific embodiment four: The difference between this embodiment and specific embodiment two is that the organic layer drying in step one uses anhydrous sodium sulfate.

[0069] Specific embodiment five: The difference between this embodiment and specific embodiment two is that the product extraction in step one uses dichloromethane.

[0070] Specific embodiment six: The difference between this embodiment and specific embodiment two is that the mass fraction of the sodium carbonate aqueous solution in step three is 30-40%.

[0071] Specific embodiment seven: The difference between this embodiment and specific embodiment two is that the purification in step three uses silica gel column chromatography.

[0072] Specific embodiment eight: The difference between this embodiment and specific embodiment two is that the recrystallization in step five uses o-dimethylbenzene.

[0073] Specific embodiment nine: The difference between this embodiment and specific embodiment two is that the rare earth triflate in step six is Eu (OTf) 3.

[0074] Specific embodiment ten: The chiral amino acid CPL probe based on aldehyde functionalized luminescent rare earth helix complex is used for detecting thiol amino acids.

[0075] 1. Excellent water solubility and physiological adaptability: Existing lanthanide helical complexes generally suffer from poor water solubility, limiting their application in biomolecular detection. This embodiment forms a hemiacetal structure through the aldehyde group, enabling the probe to achieve excellent water solubility at 3 × 10⁻⁶ ppm. -5 ~2.5×10 -4 It exhibits good water solubility within the M concentration range and strong stability in physiological buffer systems with pH 6.8–8.2 (no significant changes in luminescence intensity and spectral profile), making it directly applicable to detection scenarios in aqueous solutions and physiological environments.

[0076] 2. High Selectivity: Existing amino acid detection probes often face the problem of insufficient selectivity due to the structural similarity of amino acids, making it difficult to distinguish between thiol-containing and non-thiol-containing amino acids and their corresponding enantiomers. This embodiment utilizes the reaction mechanism of aldehyde groups specifically forming thiazolidinyl rings with thiol-containing amino acids, generating CPL responses only for D-penicillamine (D-PA) and L-cysteine ​​(L-Cys), while showing no obvious signal for the other 6 common amino acids; furthermore, D-PA induces a negative signal (g... lum =-0.23), L-Cys induced positive signal (g lum =+0.04), to achieve accurate differentiation of enantiomers.

[0077] 3. Highly sensitive CPL signal and excellent quantitative performance: Some existing CPL probes suffer from weak signals, narrow quantitative ranges, or high detection limits. This embodiment provides a rapid CPL signal response for D-PA, stabilizing within 30 minutes, and maintaining a range of 0~0.8×10⁻⁶. -3 M shows a good linear relationship within the concentration range (R 2 =0.99174), with a detection limit as low as 1.08×10⁻⁶. -5 M is far superior to existing similar detection methods and can meet the need for accurate quantification of trace amounts of thiol-containing amino acids.

[0078] 4. Strong anti-interference ability and high practical application value: Existing probes are easily interfered with by other biomolecules in complex matrices and are difficult to apply directly to the detection of actual samples. This embodiment can maintain a high intensity of the detection signal of D-PA even in the presence of the other 7 amino acids at equivalent concentrations, demonstrating outstanding anti-interference performance; at the same time, it has been successfully applied to the determination of D-PA content in commercial penicillamine tablets, with a recovery rate of 96.67%~100.01% and a relative standard deviation (RSD) ≤2.3%, requiring no complicated sample pretreatment and possessing strong practicality.

[0079] 5、The functionalization strategy is universal and expandable: The functionalization modification of existing water-soluble probes mainly focuses on improving solubility, and lacks the cooperative design of specific recognition sites. The aldehyde functionalization strategy proposed in this embodiment not only solves the water solubility problem of lanthanide helix complexes through hemiacetal structure, but also realizes targeted recognition with aldehyde group as specific reaction site, providing a universal paradigm for designing water-soluble coordination supramolecular probes for amine-containing biomolecules, and expanding the application scenarios of lanthanide helix complexes in the field of chiral biosensing.

[0080] Example 1

[0081] The structural formula of the chiral amino acid CPL probe based on the aldehyde functionalized luminescent rare earth helix complex is as follows:

[0082] ;

[0083] The structural formula of the rare earth helix is (HNEt3)2[Eu2L4], and the structural formula of the ligand L is:

[0084] .

[0085] The preparation method of the chiral amino acid CPL probe based on the aldehyde functionalized luminescent rare earth helix complex is carried out according to the following steps:

[0086] Step one, synthesis of 4-hydroxy-3-iodoacetophenone

[0087] 5 g of 4-hydroxyacetophenone, 4.66 g of elemental iodine and 11.5 mL of hydrogen peroxide aqueous solution were added to 150 mL of water, stirred at room temperature for 24 h, filtered to obtain a solid, then recrystallized by ethanol to obtain 4-hydroxy-3-iodoacetophenone as white crystals; the yield is 72%.

[0088] The mass fraction of the hydrogen peroxide aqueous solution is 30%;

[0089] Step two, synthesis of 1-(3-iodo-4-(2,2-dimethoxyethoxy) phenyl) ethanone

[0090] In a Schlenk flask, 5 g of 4-hydroxy-3-iodoacetophenone and 9.33 g of Cs2CO3 were added under N2 atmosphere, and then 150 mL of N,N-dimethylacetamide was added, and stirred at 110°C for 0.5 h; then 6.45 g of 2-bromo-1,1-dimethoxyethane was added and reacted for 5 h; the product was poured into water for quenching and extracted with dichloromethane; the obtained organic layers were combined and washed with saturated sodium chloride solution; the organic layer was dried with anhydrous sodium sulfate, and then concentrated to remove the solvent; finally, purification was performed by silica gel column chromatography to obtain 1-(3-iodo-4-(2,2-dimethoxyethoxy)phenyl)ethanone as a white crystal; the yield was 75%;

[0091] Step three, synthesis of 5-acetyl-2-(2,2-dimethoxyethoxy)-4'-acetylbiphenyl

[0092] In a Schlenk flask, 2 g of 1-(3-iodo-4-(2,2-dimethoxyethoxy)phenyl)ethanone, 0.33 g of Pd(PPh3)4 and 1.12 g of 4-acetylphenylboronic acid were placed under N2 atmosphere; 50 mL of tetrahydrofuran and 25 mL of aqueous sodium carbonate solution (30%) were sequentially added, and then stirred under reflux; after 12 h, the reaction solution was poured into water and extracted with ethyl acetate; the organic phases were combined and washed with saturated aqueous sodium chloride solution; the organic phase was dried with anhydrous sodium sulfate, and then concentrated to remove the solvent; finally, purification was performed by silica gel column chromatography to obtain 5-acetyl-2-(2,2-dimethoxyethoxy)-4'-acetylbiphenyl as a white solid; the yield was 70%.

[0093] The mass fraction of the aqueous sodium carbonate solution was 30%;

[0094] Step four, synthesis of 5-(3-oxo-4,4,4-trifluorobutenyl)-2-(2,2-dimethoxyethoxy)-4'-(3-oxo-4,4,4-trifluorobutenyl)biphenyl

[0095] In 20 mL of dimethyl ether, 1.26 g of sodium methoxide and 3.32 g of ethyl trifluoroacetate were dissolved, respectively; finally, 1 g of 5-acetyl-2-(2,2-dimethoxyethoxy)-4'-acetylbiphenyl was added, and stirred at room temperature for 24 h; the reaction solution was poured into water, and the pH was adjusted to 3 with HCl; the solid was precipitated and filtered under reduced pressure, and dried to obtain 5-(3-oxo-4,4,4-trifluorobutenyl)-2-(2,2-dimethoxyethoxy)-4'-(3-oxo-4,4,4-trifluorobutenyl)biphenyl as a white solid; the yield was 85%.

[0096] Step five, synthesis of ligand L:

[0097] The 0.5 g 5-(3-oxo-4,4,4-trifluorobutenyl)-2-(2,2-dimethoxyethoxy)-4'-(3-oxo-4,4,4-trifluorobutenyl) biphenyl was dissolved in 10 mL 1,4-dioxane, 1 mL hydrochloric acid was added and refluxed for 40 min; after the reaction was completed, it was poured into 10 mL ice water, filtered under reduced pressure to obtain a solid, washed with water until pH = 7, and finally recrystallized with o-xylene to obtain the ligand L as a white solid; the yield was 90%.

[0098] Step six, synthesis of (HNEt3)2[Eu2L4]

[0099] The 0.3 g ligand L and 0.2 g triethylamine were dissolved in 10 mL methanol to obtain a reaction solution, which was stirred until the reaction solution was clear; a rare earth triflate salt was dissolved in 3 mL methanol at room temperature to obtain a rare earth salt solution; the rare earth salt solution was added to the reaction solution, and after the reaction solution was completely clear, stirring was continued for 12 hours, then the obtained reaction solution was added to 10 mL water to form a flocculent precipitate, which was filtered after standing to obtain (HNEt3)2[Eu2L4]; Et is ethyl;

[0100] The rare earth triflate salt is Eu (OTf)3.

[0101] This embodiment is based on the chiral amino acid CPL probe of aldehyde group functionalized luminescent rare earth helicate complex for detecting thiol amino acid.

[0102] Table 1 is the crystallographic parameters of the achiral rare earth supramolecular helicate crystal (HNEt3)2[Eu2L4] prepared in Example 1.

[0103] Table 1

[0104]

[0105] The (HNEt3)2[Eu2L4] prepared in this embodiment (hereinafter referred to as the complex) was detected as follows:

[0106] 2.5 mg of the complex was dissolved in 1 mL of CH3OH, and then 9 mL of an aqueous HEPES buffer solution (pH adjusted to 7.7) was added to prepare a complex mother liquor with a concentration of 10 -4 mol / L (HEPES is used to maintain the pH stability of the system and reduce interference); 8 kinds of chiral amino acids were respectively prepared into aqueous solutions, and 40 eq of each was added into the above mother liquor in turn, and reacted for 0.5 h at constant temperature; the reaction solution was injected into a 1 mm light path quartz cuvette, 375 nm laser was used as the excitation light source, and the emission spectrum data was collected in the wavelength range of 570-600 nm. Figure 1Emission spectra of the complex in CH3OH / HEPES solution in the presence of 40 equivalents of different chiral amino acids.

[0107] Take 2.6 mg of the complex dissolved in 1 mL of CH3OH, then add 9 mL of 0.1 mol / L HEPES aqueous buffer solution (pH adjusted to 7.7), to prepare a complex stock solution with a concentration of 10 -4 mol / L, CH3OH:H2O volume ratio 1:9; 8 kinds of chiral amino acids are respectively prepared into aqueous solution, and 40 eq. are added to the above stock solution in turn (0.5 mL), and the reaction is kept constant for 0.5 h; take the reaction solution and inject it into a 1 mm light path quartz cuvette, take 375 nm laser as excitation light source, collect CPL spectrum data (use |ΔI| / |ΔI0| data for plotting) in the wavelength range of 570-600 nm. Figure 2 CPL spectra of the complex in CH3OH / HEPES buffer in the presence of 40 equivalents of different chiral amino acids.

[0108] Take 2.6 mg of the complex dissolved in 1 mL of CH3OH, then add 9 mL of 0.1 mol / L HEPES aqueous buffer solution (pH adjusted to 7.7), to prepare a complex stock solution with a concentration of 10 -4 mol / L, CH3OH:H2O volume ratio 1:9; 8 kinds of chiral amino acids are respectively prepared into aqueous solution, and 40 eq. are added to the above stock solution in turn (0.5 mL), and the reaction is kept constant for 0.5 h; take the reaction solution and inject it into a 1 mm light path quartz cuvette, take 375 nm laser as excitation light source, collect CPL spectrum data (use |ΔI| / |ΔI0| data for plotting) in the wavelength range of 570-600 nm. Figure 3 Emission enhancement ratio (|ΔI| / |ΔI0|) of the complex to different chiral amino acids.

[0109] First, 40 eq. of D-PA was added to 0.5 mL of CH3OH / HEPES buffer (v / v = 1:9, pH = 7.7, concentration = 1.0 × 10 -4 M) and reacted for 0.5 h, then the other seven kinds of amino acids (40 eq. each) were added and reacted for 0.5 h, and the change in the luminescence asymmetry factor (g lum ) was monitored by CPL spectrum. Figure 4 The g lum of the complex containing 40 eq. of D-penicillamine (D-PA) in CH3OH / HEPES buffer (yellow column) and the change in the g lum of the complex after adding 40 eq. of different chiral amino acids (green column).

[0110] From Figures 1-4It was observed that the peak shape and intensity of the emission (PL) spectrum remained almost unchanged after adding 40 equivalents of each amino acid. This indicates that the helical complex cannot serve as an effective amino acid sensor based on PL technology. In contrast, the complex exhibited selective CPL responses to the thiol-containing amino acids D-penicillamine (D-PA) and L-cysteine ​​(L-Cys). The addition of D-PA and L-Cys resulted in peak shapes and intensities in the emission (PL) spectrum. 5 D0→ 7 F1 transition (emission wavelength λ) em Negative and positive CPL signals were induced at a wavelength of 584-600 nm, while the other six amino acids did not produce detectable CPL signals. The luminescence asymmetry factor (g...) lum The definition of g is... lum = 2(I L - I R ) / (I L + I R ), where I L and I R These represent the left-handed and right-handed circularly polarized emission intensities, respectively (within the range of −2≤g). lum ≤2), is a key parameter for quantifying the degree of CPL. D-PA corresponds to g lum The value is -0.23, while the corresponding g for L-Cys is... lum The value was relatively small, at +0.04. Based on the changes in the CPL signal, the anti-interference ability of this probe to detect D-PA was further investigated through a competitive binding experiment. Under the condition that the concentrations of the other seven amino acids were equal to those of D-PA, except in the presence of the thiol-containing amino acid L-Cys, g... lum Except when the value drops to 78% of its maximum value (-0.23), g remains constant. lum The values ​​remained at 91%–96% of the maximum value. These results indicate that the probe is significantly more selective for thiol-containing amino acids than for non-thiol-containing amino acids, and can distinguish between D-PA and L-Cys based on the directional differences in the CPL response.

[0111] Dissolve 2.5 mg of the complex in 1 mL of CH3OH, then add 9 mL of HEPES buffer solution (pH adjusted to 7.7) to prepare a concentration of 10. -4 Prepare a mol / L mother liquor of the complex; prepare an aqueous solution of D-penicillamine (D-PA) and add it to the above mother liquor in 40 eq increments, and record the reaction time (0-40 min); inject the reaction solution into a 1 mm optical path quartz cuvette, and collect CPL spectral data in the wavelength range of 570-600 nm using a 375 nm laser as the excitation source. Figure 5Time-dependent CPL spectra of the complex after the addition of 0.8 × 10⁻³ M D-penicillamine (D-PA) to CH3OH / HEPES solution. Figure 6 The complex was added to a CH3OH / HEPES solution at a concentration of 0.8 × 10⁻⁶. -3 The time curve of CPL signal intensity change (ΔI) at 588 nm after MD-penicillamine (D-PA).

[0112] Dissolve 2.5 mg of the complex in 1 mL of CH3OH, then add 9 mL of 0.1 mol / L HEPES buffer solution (adjusted to pH 7.7) to prepare a solution with 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. D-penicillamine (D-PA) was prepared into an aqueous solution, and seven different concentrations of aqueous solutions (0-1.2 mM) were prepared and added to the above mother liquor in sequence. The reaction was carried out at a constant temperature for 0.5 h. 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 7 CPL response of the complex to 0-1.2 mM D-penicillamine (D-PA) in CH3OH / HEPES solution after 30 min of reaction. Figure 8 The linear fitting relationship between the signal intensity change (ΔI) of the complex in CH3OH / HEPES solution for 0-1.2 mM D-penicillamine (D-PA) at 588 nm after 30 min of reaction and the concentration of D-PA.

[0113] Depend on Figures 5-8 It can be seen that the difference in emission intensity between left-handed and right-handed circularly polarized light, ΔI = I L - I R To achieve accurate quantitative detection of D-PA concentration, the probe response time was first examined: The probe was added to a complex solution (methanol / HEPES buffer, volume ratio 1:9, pH=7.7, concentration c=1.0×10⁻⁶). -4 Add 0.8 × 10 to M) -3 D-PA at M was monitored at 5-minute intervals. The ΔI value at 588 nm rose rapidly, reaching a stable plateau after 30 minutes. Therefore, 30 minutes was set as the optimal reaction time for all subsequent quantitative analyses. When the D-PA concentration was between 0 and 1.2 × 10⁻⁶, the reaction was successful. -3 When the value of ΔI at 588 nm varies within the range of M, it shows a gradual increasing trend. Further analysis of the relationship between the ΔI value at 588 nm and the D-PA concentration revealed that the two values ​​range from 0 to 0.8 × 10⁻⁶. -3M showed good linear correlation in the range. The limit of detection of this method was calculated to be 1.08 x 10 -5 M (signal-to-noise ratio S / N = 3), further highlighting the detection capability of this probe for trace amounts of D-PA.

Claims

1. A chiral amino acid CPL probe based on aldehyde group functionalized luminescent rare earth helicate complex, characterized in that: The structural formula of the chiral amino acid CPL probe based on aldehyde group functionalized luminescent rare earth helicate complex is: ； The structural general formula of the rare earth helicate is (HNEt3)2[Eu2L4], and the structural formula of the ligand L is: 。 2. The method for preparing a chiral amino acid CPL probe based on aldehyde group functionalized luminescent rare earth helicate complex according to claim 1, characterized in that: The method is performed according to the following steps: Step one, synthesis of 4-hydroxy-3-iodoacetophenone 3~5 g 4-hydroxyacetophenone, 4~6 g iodine and 10~20 mL hydrogen peroxide aqueous solution are added into 100~200 mL water, stirred at room temperature for 24 h, filtered to obtain a solid, and then recrystallized by ethanol to obtain 4-hydroxy-3-iodoacetophenone; Step two, synthesis of 1-(3-iodo-4-(2,2-dimethoxyethoxy) phenyl) ethanone 4~6 g 4-hydroxy-3-iodoacetophenone and 8~10 g Cs2CO3 are added into a Schlenk flask under N2 atmosphere, then 100~200 mL N,N-dimethylacetamide is added, stirred at 110 ℃ for 0.5 h; then 5~7 g 2-bromo-1,1-dimethoxyethane is added and reacted for 5 h; the product is poured into water for quenching and product extraction; the obtained organic layer is washed with saturated sodium chloride solution; after the organic layer is dried, the solvent is removed by concentration; finally, purification is performed by silica gel column chromatography to obtain 1-(3-iodo-4-(2,2-dimethoxyethoxy) phenyl) ethanone; Step three, synthesis of 5-acetyl-2-(2,2-dimethoxyethoxy)-4'-acetylbiphenyl 1~3 g 1-(3-iodo-4-(2,2-dimethoxyethoxy) phenyl) ethanone, 0.2~0.4 g Pd(PPh3)4 and 1~2 g 4-acetylphenylboronic acid are placed in a Schlenk flask under N2 atmosphere; 40~60 mL tetrahydrofuran and 20~30 mL sodium carbonate aqueous solution are added in sequence, and then reflux stirring is performed, after 12 h, the reaction solution is poured into water and extracted with ethyl acetate; the combined organic phase is washed with saturated sodium chloride aqueous solution, and the organic phase is dried with anhydrous sodium sulfate, then the solvent is removed by concentration; finally, purification is performed to obtain 5-acetyl-2-(2,2-dimethoxyethoxy)-4'-acetylbiphenyl; Step four, synthesis of 5-(3-oxo-4,4,4-trifluorobutenyl)-2-(2,2-dimethoxyethoxy)-4'-(3-oxo-4,4,4-trifluorobutenyl) biphenyl 1~3 g sodium methoxide and 3~5 g ethyl trifluoroacetate are respectively dissolved in 10~30 mL dimethyl ether; finally, 1~3 g 5-acetyl-2-(2,2-dimethoxyethoxy)-4'-acetylbiphenyl is added, and stirred at room temperature for 24 h; the reaction solution is poured into water, the pH is adjusted to 2~3 with HCl, the solid is precipitated, and then dried by filtration under reduced pressure to obtain 5-(3-oxo-4,4,4-trifluorobutenyl)-2-(2,2-dimethoxyethoxy)-4'-(3-oxo-4,4,4-trifluorobutenyl) biphenyl; Step five, synthesis of ligand L: Dissolve 0.1-0.5 g 5-(3-oxo-4,4,4-trifluorobutenyl)-2-(2,2-dimethoxyethoxy)-4'-(3-oxo-4,4,4-trifluorobutenyl) biphenyl in 10-30 mL 1,4-dioxane, add 1-3 mL hydrochloric acid and reflux for 40 min; after the reaction is completed, pour into 10 mL ice water, filter under reduced pressure to obtain a solid, wash with water until pH = 7, and finally recrystallize to obtain the ligand L; Step six, synthesis of (HNEt3)2[Eu2L4] Dissolve 0.3-0.6 g of the ligand L and 0.2-0.4 g of triethylamine in 10-20 mL of methanol to obtain a reaction solution, and stir until the reaction solution is clear; dissolve a rare earth triflate in 3-5 mL of methanol to obtain a rare earth salt solution under room temperature conditions; add the rare earth salt solution to the reaction solution, continue to stir after the reaction solution is completely clear for 12-24 hours, then add the obtained reaction solution to 10-20 mL of water to form a flocculent precipitate, and after standing, filter to obtain (HNEt3)2[Eu2L4].

3. The method for preparing a chiral amino acid CPL probe based on aldehyde group functionalized luminescent rare earth helicate complex according to claim 2, characterized in that: The mass fraction of the hydrogen peroxide aqueous solution in step one is 30%.

4. The method for preparing a chiral amino acid CPL probe based on aldehyde group functionalized luminescent rare earth helicate complex according to claim 2, characterized in that: The organic layer drying in step one uses anhydrous sodium sulfate.

5. The method for preparing a chiral amino acid CPL probe based on aldehyde group functionalized luminescent rare earth helicate complex according to claim 2, characterized in that: The product extraction in step one uses dichloromethane.

6. The method for preparing a chiral amino acid CPL probe based on aldehyde group functionalized luminescent rare earth helicate complex according to claim 2, characterized in that: The mass fraction of the sodium carbonate aqueous solution in step three is 30-40%.

7. The method for preparing a chiral amino acid CPL probe based on aldehyde group functionalized luminescent rare earth helicate complex according to claim 2, characterized in that: The purification in step three uses a silica gel column chromatography method.

8. The method for preparing a chiral amino acid CPL probe based on aldehyde group functionalized luminescent rare earth helicate complex according to claim 2, characterized in that: The recrystallization in step five uses o-xylene.

9. The method for preparing a chiral amino acid CPL probe based on aldehyde group functionalized luminescent rare earth helicate complex according to claim 2, characterized in that: The rare earth triflate in step six is Eu (OTf)3.

10. Use of a chiral amino acid CPL probe based on aldehyde functionalized luminescent rare earth helicate complexes according to claim 1, characterized in that: A chiral amino acid CPL probe based on aldehyde group functionalized luminescent rare earth helicate complexes is used for detecting sulfhydryl amino acids.

Images