Preparation and detection method of fluorescent probe for D-carnitine
By preparing fluorescent probes (S,S)-4 and conducting fluorescence tests, the problem of expensive and complex operation of D-carnitine detection equipment in the prior art is solved, and high sensitivity and low cost detection of D-carnitine is achieved.
Patent Information
- Application Number
- CN202510754332.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-06
- Publication Date
- 2025-08-26
AI Technical Summary
The prior art is difficult to detect D-carnitine quickly, easily and with high sensitivity. The traditional methods and equipment are expensive, cumbersome, and have low sensitivity, making it difficult to meet the needs of medical diagnosis and physiological research.
A fluorescent probe is prepared, (S,S)-4 fluorescent probe is synthesized through specific chemical steps, and reacted with D-carnitine and zinc ions for fluorescence testing under specific conditions to achieve enantioselective recognition of D-carnitine.
The distinction between D-carnitine and L-carnitine in real human semen is achieved. The detection method is simple, low-cost, high sensitivity and a wide range of detection concentrations.
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Figure CN120535459A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of chemical analysis and detection, and in particular to a preparation and detection method of a fluorescent probe targeting D-carnitine. Background Art
[0002] Carnitine is a quaternary ammonium compound widely found in organisms. Its D- and L-forms have distinct physiological functions. L-carnitine is a key cofactor for fatty acid β-oxidation in mammalian cells, participating in energy metabolism by transporting long-chain fatty acids to the mitochondrial matrix. It is particularly highly concentrated in cardiac, skeletal muscle, and testicular tissues. In contrast, D-carnitine, as a non-physiological enantiomer of L-carnitine, not only cannot participate in fatty acid metabolism but can also disrupt normal metabolic pathways by competitively inhibiting the L-carnitine transporter (OCTN2). Notably, D-carnitine is virtually undetectable in healthy individuals, but under certain pathological conditions, its abnormal accumulation may exacerbate reproductive system damage.
[0003] Currently, the detection of D-carnitine primarily relies on chiral HPLC or mass spectrometry. These methods suffer from expensive instrumentation, cumbersome operation, low sensitivity, and long detection cycles, making them difficult to meet the demands of rapid screening. Fluorescence sensing, on the other hand, offers advantages such as high selectivity, high sensitivity, low detection limits, and ease of use. Therefore, the development of simple, sensitive, and efficient detection and analysis technologies for D-carnitine is urgently needed in the fields of medical diagnosis, physiological research, and nutritional science. Summary of the Invention
[0004] The purpose of the present invention is to provide a preparation and detection method for a fluorescent probe for D-carnitine, which can selectively identify the structure of the D-carnitine fluorescent probe, so as to achieve the enantioselective recognition of D-carnitine and L-carnitine by the fluorescent probe in real human semen, thereby making it have the advantages of enantioselectivity, high sensitivity, and a wide detection concentration range.
[0005] The above technical objectives of the present invention are achieved through the following technical solutions: A fluorescent probe for detecting D-carnitine, the specific chemical structure is shown below: .
[0006] The present invention also provides a method for preparing a fluorescent probe for D-carnitine, comprising the following steps: Step 1: Weigh (S)-1,1'-bi-2,2'-naphthol and dissolve it in an appropriate amount of anhydrous dichloromethane. Stir thoroughly to dissolve it. Then, add N,N-diisopropylethylamine at 0°C and stir thoroughly to dissolve it for 3 hours. Next, add bromomethyl methyl ether and react at 0°C for 1 hour. After the reaction is completed, hydrochloric acid is added to quench the reaction, the reaction solution is extracted with dichloromethane and dried over anhydrous sodium sulfate, the reaction solvent is removed by rotary evaporation, and the crude product is purified by column chromatography and eluted with petroleum ether / ethyl acetate to obtain a white solid product (S)-1; Step 2: Weigh the intermediate (S)-1 and dissolve it in an appropriate amount of tetrahydrofuran. After the raw materials are fully stirred and dissolved and the temperature is reduced to 0°C, n-butyl lithium solution is slowly added dropwise. The temperature is naturally restored to room temperature and the reaction is carried out for 2 hours. After the temperature is reduced to 0°C, anhydrous N,N-dimethylformamide is added and the reaction is carried out for 3 hours. After the reaction is completed, saturated ammonium chloride solution is added to quench the reaction, the reaction solution is extracted with ethyl acetate and dried over anhydrous sodium sulfate, the reaction solvent is removed by rotary evaporation, and the crude product is purified by column chromatography, eluted with petroleum ether / ethyl acetate, and filtered to obtain a yellow solid product (S)-2; Step 3: Weigh the intermediate (S)-2 and dissolve it in an appropriate amount of anhydrous N,N-dimethylformamide. Add K2CO3 and heat under reflux at 80°C for 3 hours. After the reaction is completed, dissolve 2,6-bis-(bromomethyl)pyridine in N,N-dimethylformamide and add it dropwise to the reaction solution. Heat the reaction overnight. After overnight reaction, the reaction solution was poured into an appropriate amount of ultrapure water, extracted with ethyl acetate, washed with ultrapure water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and the reaction solvent was removed by rotary evaporation. The crude product was purified by column chromatography and eluted with petroleum ether / ethyl acetate to obtain a yellow product (S, S)-3; Step 4: Weigh the intermediate (S, S)-3 and dissolve it in appropriate amounts of dichloromethane and ethanol. Add concentrated hydrochloric acid and react overnight. After overnight reaction, add solid sodium bicarbonate to quench the reaction until no bubbles are generated on the surface of the reaction solution. Extract with dichloromethane and wash with saturated sodium chloride solution. Dry with anhydrous sodium sulfate and filter to remove the solvent to obtain a yellow solid product (S, S)-4.
[0007] Further: the reactions in steps 1-3 are all carried out under nitrogen protection.
[0008] Further: in step 1, the volume ratio of the elution phase for column chromatography purification is petroleum ether:ethyl acetate=40:1.
[0009] Furthermore: in step 2, n-BuLi needs to be slowly added dropwise while stirring, and the volume ratio of the elution phase for column chromatography purification is petroleum ether:ethyl acetate = 10:1.
[0010] Furthermore: in step 3, the volume ratio of the elution phase of the column chromatography purification is petroleum ether:ethyl acetate=6:1, and the molar ratio of the (S)-2:2,6-bis-(bromomethyl)pyridine is 2.3:1.3.
[0011] Further: in step 4, calcium bicarbonate solid powder is added in small amounts and multiple times to quench the reaction solution until no bubbles are generated on the surface of the reaction solution.
[0012] The present invention also provides a detection method for a fluorescent probe targeting D-carnitine, which is characterized by: dissolving a fluorescent probe of an appropriate concentration in an appropriate amount of chromatographic grade dimethyl sulfoxide to prepare a concentration probe solution; then, adding N,N-dimethylformamide solution, probe solution, carnitine solution and zinc ion solution in sequence to a 5 ml centrifuge tube, reacting for a period of time, and then adding a certain volume of N,N-dimethylformamide to dilute and fix the volume, and performing fluorescence testing.
[0013] Furthermore: the optimal test condition of the fluorescent probe is a molar ratio of fluorescent probe: carnitine: zinc ion of 1.6:32:3.2.
[0014] In summary, the present invention has the following beneficial effects: First, the fluorescent probe of the present invention is simple to prepare and low in cost, and can distinguish the two configurations of carnitine in real human seminal plasma; Secondly, the detection method provided by the present invention is simple to operate, uses low-cost solvents, and has convenient post-processing. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1 This is the synthetic route of the fluorescent probe (S,S)-4 of the present invention; Figure 2 (a) is the H NMR spectrum of (S,S)-4; (b) is the C NMR spectrum of (S,S)-4; Figure 3 This is the solvent screening test diagram of (S,S)-4; Figure 4 (a) is a fluorescence spectrum diagram of the fluorescent probe (S,S)-4 and D / L-carnitine at different concentrations of the present invention; (b) is a fluorescence spectrum trend diagram of (S,S)-4 and D / L-carnitine at different concentrations; Figure 5 (a) is the fluorescence spectra of (S,S)-4 after reaction with D / L-carnitine at different times; (b) is the fluorescence spectrum trend diagram of (S,S)-4 and D / L-carnitine at different times; Figure 6 These are the fluorescence spectra of (S,S)-4 and D / L-carnitine under the action of different concentrations of zinc ions. DETAILED DESCRIPTION
[0016] The present invention will be further described in detail below with reference to the accompanying drawings.
[0017] In the description of the present invention, it should be understood that the terms "up", "down", "left", "right", "front", "back", "inside", "outside", etc., indicating directions or positional relationships, are based on the directions or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific direction, be constructed and operated in a specific direction, and therefore cannot be understood as a limitation on the present invention.
[0018] Example 1, with reference to Figure 1-2 A method for preparing and detecting a fluorescent probe for D-carnitine comprises the following steps: Step 1: Weigh 5 g (17.5 mmol) of (S)-1,1'-bi-2,2'-naphthol and dissolve it in 100 mL of anhydrous dichloromethane. Stir thoroughly to dissolve. Then, add 6.4 mL (38.5 mmol) of N,N-diisopropylethylamine at 0°C and stir thoroughly for 3 hours. Next, add 2.1 mL (26.2 mmol) of bromomethyl methyl ether at 0°C and react for 1 hour. After the reaction was complete, 50 mL of 2 M hydrochloric acid was added to quench the reaction. The reaction solution was extracted with dichloromethane and dried over anhydrous sodium sulfate. The reaction solvent was removed by rotary evaporation, and the crude product was purified by column chromatography using petroleum ether / ethyl acetate as the eluent to obtain the white solid product (S)-1. This operation was carried out under nitrogen protection throughout. Step 2: Weigh 1 g, 3 mmol of intermediate (S)-1 and dissolve it in 60 mL of tetrahydrofuran. After the raw materials are fully stirred and dissolved and the temperature is lowered to 0°C, 6.6 mL, 10.5 mmol of n-butyl lithium solution is slowly added dropwise and the temperature is naturally returned to room temperature to react for 2 hours. After the temperature was lowered to 0°C again, 0.35 mL and 4.5 mmol of anhydrous N,N-dimethylformamide were added and the reaction was allowed to proceed for 3 hours. After completion of the reaction, saturated ammonium chloride solution was added to quench the reaction. The reaction solution was extracted with ethyl acetate and dried over anhydrous sodium sulfate. The reaction solvent was removed by rotary evaporation, and the crude product was purified by column chromatography, eluted with petroleum ether / ethyl acetate, and filtered to obtain a yellow solid product (S)-2. The entire operation was carried out under nitrogen protection. Step 3: Weigh 0.8 g, 2.3 mmol of intermediate (S)-2 and dissolve it in 6 mL of anhydrous N,N-dimethylformamide. Then add 0.774 g, 5.6 mmol of K2CO3 and heat under reflux at 80°C for 3 hours. After the reaction is completed, dissolve 0.332 g, 1.3 mmol of 2,6-bis-(bromomethyl)pyridine in 1 mL of N,N-dimethylformamide and add it dropwise to the reaction solution. Heat the reaction overnight. After overnight reaction, the reaction solution was poured into 100 mL of ultrapure water, extracted with ethyl acetate, washed with ultrapure water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and the reaction solvent was removed by rotary evaporation. The crude product was purified by column chromatography using a ratio of 3:1 petroleum ether:ethyl acetate, and eluted with petroleum ether / ethyl acetate to obtain a yellow product (S,S)-3. The entire operation was carried out under nitrogen protection. Step 4: Weigh 0.2 g, 0.24 mmol of the intermediate (S, S)-3 and dissolve it in 30 mL of dichloromethane and 30 mL of ethanol. Add 12 N concentrated hydrochloric acid and react overnight. After overnight reaction, add solid sodium bicarbonate to quench the reaction until no bubbles are generated on the surface of the reaction solution. Extract with dichloromethane and wash with saturated sodium chloride solution. Dry with anhydrous sodium sulfate and filter to remove the solvent to obtain a yellow solid product (S, S)-4.
[0019] Example 2, a method for detecting a fluorescent probe for D-carnitine, wherein the probe (S,S)-4 is dissolved in an appropriate amount of chromatographic grade N,N-dimethylformamide to prepare a 1.6 mM probe solution, and then carnitine and zinc ions are dissolved in ultrapure water for later use. Subsequently, 250 ul of N,N-dimethylformamide solution, 50 ul of probe solution, 50 ul of carnitine solution, and 50 ul of zinc ion solution are added to a 5 mL centrifuge tube in sequence, wherein the molar ratio of fluorescent probe: carnitine: zinc ion is 1.6:32:3.2. After reacting for 3 hours, 3.6 ml of N,N-dimethylformamide solution is added for fluorescence testing, and the fluorescence test parameters are controlled as follows: excitation light wavelength is 10 nm, emission light slit is 10 nm, and excitation light wavelength is 440 nm.
[0020] Reference Figure 3 The fluorescent probe (S, S)-4 was reacted with two different configurations of carnitine in six solvents, and only D-configuration carnitine showed enantioselectivity in DMF and DMSO solutions, and only D-configuration carnitine showed obvious fluorescence enhancement; Reference Figure 4 When the fluorescent probe (S, S)-4 was reacted with two different configurations of carnitine at different concentrations, the fluorescence intensity reached the highest value when the carnitine equivalent was 20eq and showed excellent enantioselectivity; Reference Figure 5 ,When the fluorescent probe (S, S)-4 was treated with two different configurations of carnitine at different times, the fluorescence intensity reached a plateau at 3 h; Reference Figure 6 When the fluorescent probe (S, S)-4 was used to react with two different configurations of carnitine and different concentrations of zinc ions, the best enantioselectivity was shown when the zinc ion equivalent was 2 equivalents; The fluorescent probe of the present invention can distinguish between D- and L-configuration carnitine in N,N-dimethylformamide.
[0021] The detection method provided by the present invention is simple to operate, uses a low-cost solvent, and has convenient post-processing.
[0022] This specific embodiment is merely an explanation of the present invention and is not intended to limit the present invention. After reading this specification, those skilled in the art may make creative modifications to this embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by patent law.
Claims
1. A fluorescent probe for detecting D-carnitine, characterized in that: The specific chemical structure is shown below: 。 2. The method for preparing a fluorescent probe for D-carnitine according to claim 1, wherein: The following steps are involved: Step 1: Weigh (S)-1,1'-bi-2,2'-naphthol and dissolve it in an appropriate amount of anhydrous dichloromethane. Stir thoroughly to dissolve it. Then, add N,N-diisopropylethylamine at 0°C and stir thoroughly to dissolve it for 3 hours. Next, add bromomethyl methyl ether and react at 0°C for 1 hour. After the reaction is completed, hydrochloric acid is added to quench the reaction, the reaction solution is extracted with dichloromethane and dried over anhydrous sodium sulfate, the reaction solvent is removed by rotary evaporation, and the crude product is purified by column chromatography and eluted with petroleum ether / ethyl acetate to obtain a white solid product (S)-1; Step 2: Weigh the intermediate (S)-1 and dissolve it in an appropriate amount of tetrahydrofuran. After the raw materials are fully stirred and dissolved and the temperature is reduced to 0°C, n-butyl lithium solution is slowly added dropwise. The temperature is naturally restored to room temperature and the reaction is carried out for 2 hours. After the temperature is reduced to 0°C, anhydrous N,N-dimethylformamide is added and the reaction is carried out for 3 hours. After the reaction is completed, saturated ammonium chloride solution is added to quench the reaction, the reaction solution is extracted with ethyl acetate and dried over anhydrous sodium sulfate, the reaction solvent is removed by rotary evaporation, and the crude product is purified by column chromatography, eluted with petroleum ether / ethyl acetate, and filtered to obtain a yellow solid product (S)-2; Step 3: Weigh the intermediate (S)-2 and dissolve it in an appropriate amount of anhydrous N,N-dimethylformamide. Add K2CO3 and heat under reflux at 80°C for 3 hours. After the reaction is completed, dissolve 2,6-bis-(bromomethyl)pyridine in N,N-dimethylformamide and add it dropwise to the reaction solution. Heat the reaction overnight. After overnight reaction, the reaction solution was poured into an appropriate amount of ultrapure water, extracted with ethyl acetate, washed with ultrapure water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and the reaction solvent was removed by rotary evaporation. The crude product was purified by column chromatography and eluted with petroleum ether / ethyl acetate to obtain a yellow product (S, S)-3; Step 4: Weigh the intermediate (S, S)-3 and dissolve it in appropriate amounts of dichloromethane and ethanol. Add concentrated hydrochloric acid and react overnight. After overnight reaction, add solid sodium bicarbonate to quench the reaction until no bubbles are generated on the surface of the reaction solution. Extract with dichloromethane and wash with saturated sodium chloride solution. Dry with anhydrous sodium sulfate and filter to remove the solvent to obtain a yellow solid product (S, S)-4.
3. The method for preparing a fluorescent probe for D-carnitine according to claim 2, wherein: The reactions in steps 1-3 were all carried out under nitrogen protection.
4. The method for preparing a fluorescent probe for D-carnitine according to claim 2, wherein: In step 1, the volume ratio of the elution phase for column chromatography purification is petroleum ether:ethyl acetate=40:
1.
5. The method for preparing a fluorescent probe for D-carnitine according to claim 2, wherein: In step 2, n-BuLi needs to be slowly added dropwise while stirring, and the volume ratio of the elution phase for column chromatography purification is petroleum ether:ethyl acetate = 10:
1.
6. The method for preparing a fluorescent probe for D-carnitine according to claim 2, wherein: In step 3, the volume ratio of the elution phase of the column chromatography purification is petroleum ether:ethyl acetate=6:1, and the molar ratio of the (S)-2:2,6-bis-(bromomethyl)pyridine is 2.3:1.
3.
7. The method for preparing a fluorescent probe for D-carnitine according to claim 2, wherein: In step 4, calcium bicarbonate solid powder is added in small amounts and multiple times to quench the reaction solution until no bubbles are generated on the surface of the reaction solution.
8. The method for detecting a fluorescent probe for D-carnitine according to claim 2, wherein: Dissolve the fluorescent probe of appropriate concentration in an appropriate amount of chromatographic grade dimethyl sulfoxide to prepare a concentration probe solution. Then, add N,N-dimethylformamide solution, probe solution, carnitine solution and zinc ion solution in a 5 ml centrifuge tube in sequence. After reacting for a period of time, add a certain volume of N,N-dimethylformamide to dilute the solution and perform fluorescence testing.
9. The method for preparing a fluorescent probe for D-carnitine according to claim 8, wherein: The optimal test condition for the fluorescent probe is a molar ratio of fluorescent probe: carnitine: zinc ion of 1.6:32:3.2.