A phenanthroimidazole-based fluorescent amine compound labeling reagent, its synthesis method and application

Abstract

The present invention provides a method for synthesizing a phenanthroimidazole-based fluorescent amine compound labeling reagent and its application, belonging to the technical field of fluorescent labeling of organic small molecule amine compounds. Using phenanthroimidazole as the parent ring and chloroformate as the reactive group, the chemical name is: 4-(N-phenylphenanthroimidazolyl)-benzyl chloroformate. The specific preparation method is as follows: (1) React phenanthraquinone, aniline, p-hydroxymethylbenzaldehyde, and ammonium acetate to obtain an intermediate; (2) Hydrolyze the intermediate with an aqueous potassium hydroxide solution to obtain the intermediate product 4-(N-phenylphenanthroimidazolyl)-benzyl alcohol; (3) React the intermediate product with triphosgene to obtain the target product. The present invention can quickly, accurately, and sensitively label 10 kinds of aliphatic amines, thereby realizing the separation and detection of trace aliphatic amines in complex systems, and can be used for the fluorescent labeling and analysis of various aliphatic amines in research fields such as life and food, and has broad application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of fluorescent labeling of organic small molecule amine compounds, and specifically provides a phenanthroimidazole fluorescent amine compound labeling reagent, a synthesis method and application thereof. Background Art

[0002] Most amine compounds have a pungent odor and can be harmful to human health. Many amines are not only toxic, but also easily react with nitrogen oxides and hydroxyl radicals in the air to produce strong carcinogenic substances - N-nitrosamine compounds. Therefore, establishing a highly sensitive and highly selective detection method is of great significance to protecting the environment and human health. The ideal derivatization reagent must meet the following conditions: the reagent itself must be stable and can be rapidly derivatized within a large temperature range; excess reagents or by-products do not affect separation; the reagent has strong ultraviolet absorption or emits strong fluorescence under ultraviolet light; the reagent is easy to synthesize, and the derivative has good solubility in the mobile phase system. Summary of the invention

[0003] In view of the above problems existing in the prior art, the present invention provides a phenanthroimidazole fluorescent amine compound labeling reagent with fast labeling reaction rate, high labeling product signal, high accuracy and sensitivity in fluorescent detection of amine compounds, and a synthesis method and application thereof.

[0004] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0005] On the one hand, the present invention provides a phenanthroimidazole fluorescent amine compound labeling reagent, with phenanthroimidazole as the fluorescent parent ring and chloroformate as the reactive group, and its chemical name is: 4-(N-phenylphenanthroimidazole)-benzyl chloroformate, and its chemical structure is:

[0006]

[0007] The present invention also includes a method for synthesizing a phenanthroimidazole fluorescent amine compound labeling reagent, comprising:

[0008] Step 1: reacting phenanthrenequinone, aniline, p-hydroxymethylbenzaldehyde with ammonium acetate and hydrolyzing to obtain an intermediate 4-(N-phenylphenanthroimidazolyl)-benzyl alcohol;

[0009] Step 2: react the intermediate 4-(N-phenylphenanthroimidazolyl)-benzyl alcohol with solid phosgene to obtain the target product 4-(N-phenylphenanthroimidazolyl)-benzyl chloroformate.

[0010] Furthermore, the synthesis method specifically comprises the following steps:

[0011] Step 1: Substitution reaction: Dissolve phenanthraquinone, aniline, p-hydroxymethylbenzaldehyde, and ammonium acetate in acetic acid. React under electromagnetic stirring in an oil bath at 105 °C for 4 h. After the reaction is completed, wait for the reaction solution to cool, then pour it into 400 mL of water and continuously stir to precipitate a light green solid. Filter by suction and wash twice with water. Air-dry the crude product. Dissolve the above product in ethanol, add an aqueous potassium hydroxide solution, and react under electromagnetic stirring in an oil bath at 80 °C for 1 h. After the reaction is completed, spin-dry with a rotary evaporator, and recrystallize twice with ethanol to obtain light green crystals.

[0012] Step 2: Chlorocarbonylation reaction: Dissolve triphosgene in dichloromethane. Under ice-bath cooling and stirring conditions, add a small amount of triethylamine dropwise, and gradually add dropwise a dichloromethane solution containing the intermediate 4-(N-phenylphenanthroimidazolyl)-benzyl alcohol. React under ice bath for 3 h, and then slowly raise the temperature to room temperature and react for 24 h. After the reaction is completed, filter by suction, and evaporate the filtrate to dryness under reduced pressure with a rotary evaporator, and recrystallize with dichloromethane to obtain the target product 4-(N-phenylphenanthroimidazolyl)-benzyl chloroformate.

[0013] Furthermore, in the above Step 1, the molar ratio of phenanthraquinone, aniline, p-hydroxymethylbenzaldehyde, and ammonium acetate is preferably 1:1.2:1:10, the reaction temperature is preferably 105 °C, and the reaction time is preferably 4 h; the intermediate 4-(N-phenylphenanthroimidazolyl)-benzyl alcohol is recrystallized twice with ethanol to obtain a light green crystalline product.

[0014] Furthermore, in the above Step 2, the target product 4-(N-phenylphenanthroimidazolyl)-benzyl chloroformate is recrystallized twice with dichloromethane to obtain a white needle-like crystalline product;

[0015] The molar ratio of 4-(N-phenylphenanthroimidazolyl)-benzyl chloroformate to triphosgene is preferably 1:3, and the addition amount of triethylamine is 3% of the mass of triphosgene.

[0016] The present invention also includes an application of a phenanthroimidazole-based fluorescent amine compound labeling reagent. The labeling reagent has the above structure or is synthesized by the above synthesis method, and is applied to detect the content of aliphatic amines in a sample.

[0017] Furthermore, the specific steps for detecting the content of aliphatic amines in a sample are as follows:

[0018] (1) Solution preparation: Prepare a boric acid-borax buffer solution with pH = 9.0; prepare 5 portions of mixed aliphatic amine mixed standard solutions with concentrations of 10 nM, 100 nM, 1 μM, 10 μM, and 100 μM respectively; prepare an acetonitrile solution of 4-(N-phenylphenanthroimidazolyl)-benzyl chloroformate with a concentration of 10 mM.

[0019] (2) Working curve plotting: Sequentially add 400 μL of boric acid-borax buffer solution, 20 μL of mixed fatty amine standard solution, and 100 μL of acetonitrile solution of 4-(N-phenylphenanthroimidazolyl)-benzyl chloroformate into a 2 mL ampoule. After reacting at room temperature for 4 min, the derivatized solution is processed as follows:

[0020] (a) Add 50 μL of quenching agent acetic acid to stop the reaction, and let it stand for 5 - 10 minutes for injection analysis;

[0021] (b) Take 10 μL for high performance liquid chromatography-fluorescence analysis to obtain the chromatographic separation diagram of the mixed fatty amine standard. After derivatizing and performing high performance liquid chromatography-fluorescence analysis on five portions of the mixed fatty amine standard in sequence, plot the working curve of fatty amine concentration with the fatty amine concentration as the abscissa and the peak area as the ordinate;

[0022] (3) Fatty amine concentration detection: After derivatizing and labeling the sample with 4-(N-phenylphenanthroimidazolyl)-benzyl chloroformate, perform high performance liquid chromatography-fluorescence analysis. Substitute the obtained peak area into the fatty amine concentration working curve to obtain the fatty amine concentration in the sample.

[0023] Furthermore, in the step (1), the fatty amine is a mixture of methylamine, ethylamine, propylamine, butylamine, pentylamine, hexylamine, heptylamine, octylamine, nonylamine, and decylamine.

[0024] Furthermore, the steps for detecting the fatty amine content in fresh fish and meat are as follows:

[0025] Take 1 g of fresh fish and meat, cut it into pieces and grind it in a 10 mL centrifuge tube, add 2 mL of water and 2 mL of acetonitrile, shake well and ultrasonicate for 15 min. Add 50 mg of Na 2 SO 4 to further remove the residual moisture in the food. After labeling according to the method in the step (2), perform high performance liquid chromatography-fluorescence analysis. Substitute the obtained peak area into the working curve to obtain the concentration of each fatty amine in the fresh fish and meat, and then obtain the content of each fatty amine.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The maximum excitation wavelength and emission wavelength of the reagent of the present invention are 297 nm and 418 nm respectively, and it can realize the fluorescence labeling and detection of fatty amines. This reagent uses phenanthroimidazole as the fluorescent parent ring and chloroformate as the reactive group. Its synthesis steps are simple and convenient, easy to achieve, and can be completed in three steps: (1) React phenanthraquinone, aniline, p-hydroxymethylbenzaldehyde, and ammonium acetate; (2) Hydrolyze with an alkaline solution to obtain the intermediate 4-(N-phenylphenanthroimidazolyl)-benzyl alcohol, and recrystallize twice with ethanol to obtain light green crystals; (3) React the intermediate 4-(N-phenylphenanthroimidazolyl)-benzyl alcohol with triphosgene to obtain the target product 4-(N-phenylphenanthroimidazolyl)-benzyl chloroformate, and recrystallize twice with dichloromethane to obtain a white needle-like crystalline product.

[0028] The fluorescence labeling reagent of the present invention has stable chemical properties and a chemical purity of 99.6%. The phenanthroimidazole-based fluorescence labeling reagent of the present invention uses phenanthroimidazole as the parent ring and chloroformate as the active group. When the chloroformate group labels amine compounds (such as amino acids, fatty amines), it has high selectivity for amine compounds, and the labeling time is short and the labeling yield is high. In terms of derivatization conditions, the phenanthraquinone fluorescence labeling reagent only needs to react at room temperature for 2 - 5 minutes to complete derivatization, and does not require complex extraction conditions. In terms of detection methods, high performance liquid chromatography can be used for fluorescence detection. Therefore, the chloroformate labeling reagent described in the present invention can label and detect amine compounds quickly, accurately, and sensitively, and is especially suitable for the separation and detection of trace and ultra-trace small molecule amine compounds in complex systems. It can be used for the fluorescence labeling and analysis of various fatty amines in research fields such as life analysis and food analysis, and has broad application prospects. Description of the Drawings

[0029] Figure 1 is the synthetic route diagram of preparing 4-(N-phenylphenanthroimidazolyl)-benzyl chloroformate in Example 1 of the present invention;

[0030] Figure 2 is the 1H NMR spectrum of 4-(N-phenylphenanthroimidazolyl)-benzyl chloroformate in Example 1 of the present invention;

[0031] Figure 3 is the UV-Vis absorption spectrum of 4-(N-phenylphenanthroimidazolyl)-benzyl chloroformate in Example 1 of the present invention;

[0032] Figure 4 is the fluorescence emission spectrum of 4-(N-phenylphenanthroimidazolyl)-benzyl chloroformate in Example 1 of the present invention;

[0033] Figure 5 is the reaction route diagram of the labeling reagent and fatty amine in Example 2 of the present invention;

[0034] Figure 6 It is the chromatogram of the chromatographic separation of 10 kinds of fatty amine mixed standards derived from the labeling reagent in Example 2 of the present invention;

[0035] Figure 7 It is the chromatogram of the fatty amines detected in the actual sample of fresh fish and meat by the labeling reagent in Example 3 of the present invention. Specific Embodiments

[0036] To make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following will be described in detail with specific embodiments.

[0037] The reagents and materials used in the examples and comparative examples can be obtained through commercial channels without special instructions.

[0038] The present invention provides a phenanthroimidazole-based fluorescent amine compound labeling reagent, its synthesis method and application, and the specific embodiments are as follows.

[0039] Example 1

[0040] A phenanthroimidazole-based fluorescent amine compound labeling reagent, with phenanthroimidazole as the fluorescent parent ring and chloroformate as the reactive group, its chemical name is: 4-(N-phenylphenanthroimidazolyl)-benzyl chloroformate, and its chemical structural formula is:

[0041]

[0042] The specific synthesis route is shown in Figure 1 , including:

[0043] Step 1: Substitution reaction: Add phenanthraquinone (5.0 g) and 200 mL of glacial acetic acid to a 500 mL three-necked round-bottom flask, quickly heat to 105 °C to make them miscible, and successively add the acetic acid solution of p-hydroxymethylbenzaldehyde (3.25 g), aniline (2.62 mL) and ammonium acetate (18.0 g). After heating under reflux for 4 h, cool to room temperature, slowly pour the reaction solution into 500 mL of water, and stir vigorously. Filter and collect the precipitated solid, then wash it twice with deionized water and air-dry it naturally; dissolve the solid in an ethanol solution and pour it into 20 mL of 10 M KOH aqueous solution, and heat under reflux at 80 °C for 1 h. Then rotary evaporate and concentrate the ethanol and recrystallize twice to obtain a light green crystal.

[0044] Step 2: Chlorocarbonylation reaction: Pour 200 mL of dichloromethane into a 500 mL three-necked round-bottom flask and cool it to 0 °C in an ice bath. Weigh 11.8 g of solid phosgene and pour it into the flask. React for 30 min under magnetic stirring, then add 0.5 mL of triethylamine, quickly cover the flask with a stopper, and react for 10 min. Slowly add a dichloromethane mixture solution containing 4.0 g of 4-(N-phenylphenanthroimidazolyl)-benzyl alcohol to the flask over about 1 h. After the addition is complete, react for 3 h in an ice bath, then react at room temperature for 24 h. Filter to remove the insoluble substances, evaporate the filtrate to dryness under reduced pressure using a rotary evaporator, and recrystallize twice with dichloromethane to obtain a white needle-like crystalline product.

[0045] Figure 2 is the NMR of 4-(N-phenylphenanthroimidazolyl)-benzyl chloroformate 1 1H NMR spectrum, and its characterization data are as follows: 1H NMR (500 MHz, DMSO) δ 9.03 (s, 2H), 8.97 (s, 1H), 7.90 (s, 1H), 7.85 (s, 2H), 7.75 (s, 2H), 7.72 (s, 1H), 7.69 (s, 1H), 7.66 (s, 1H), 7.64 (s, 1H), 7.44 (s, 1H), 7.41 (s, 2H), 7.05 (s, 1H), 5.76 (s, 1H), 4.54 (s, 2H).

[0046] To improve the detection sensitivity of fluorescently labeled fatty amines, accurately obtaining the maximum excitation wavelength and maximum emission wavelength of the fluorescent labeling reagent is the key to improving the detection sensitivity. The maximum excitation wavelength and maximum emission wavelength of the labeling reagent of the present invention are shown in Figures 3 - 4 , where Figure 3 is the ultraviolet-visible absorption spectrum of the labeling reagent; Figure 4 is the fluorescence emission spectrum of the labeling reagent. As can be seen from Figures 3 - 4 , the maximum excitation wavelength and maximum emission wavelength of the labeled product are 297 nm and 418 nm, respectively.

[0047] Example 2

[0048] The present invention also provides a method for detecting the content of fatty amines in a sample. Specifically:

[0049] (1) Prepare a boric acid-borax buffer solution with pH = 9.0; prepare 5 portions of mixed fatty amine mixed standard solution, where the concentration of each fatty amine (methylamine, ethylamine, propylamine, butylamine, pentylamine, hexylamine, heptylamine, octylamine, nonylamine, decylamine) is 10 nM respectively. Prepare 10 nM mixed fatty amine standard solution, and successively prepare 100 nM, 1 μM, 10 μM, and 100 μM; prepare an acetonitrile solution of 4-(N-phenylphenanthroimidazolyl)-benzyl chloroformate with a concentration of 10 mM.

[0050] (2) Add 400 μL of boric acid-borax buffer solution, 20 μL of mixed fatty amine standard solution, and 100 μL of acetonitrile solution of 4-(N-phenylphenanthroimidazolyl)-benzyl chloroformate into a 2 mL ampoule bottle in sequence. After reacting at room temperature for 4 min, the derivatization solution is processed as follows:

[0051] (a) Add 50 μL of quenching agent acetic acid to stop the reaction, and let it stand for 5 - 10 minutes for injection analysis;

[0052] (b) Take 10 μL for high performance liquid chromatography-fluorescence analysis to obtain the chromatographic separation diagram of the mixed fatty amine standard. After derivatizing and performing high performance liquid chromatography-fluorescence analysis on five portions of the mixed fatty amine standard in sequence, a working curve of fatty amine concentration is plotted with the fatty amine concentration as the abscissa and the peak area as the ordinate, as shown in Table 1;

[0053] (3) After derivatizing and labeling the sample with 4-(N-phenylphenanthroimidazolyl)-benzyl chloroformate, perform high performance liquid chromatography-fluorescence analysis. Substitute the obtained peak area into the working curve of fatty amine concentration, and the concentration of fatty amine in the sample can be obtained.

[0054] Table 1

[0055]

[0056] Note: Y is the peak area and X is the concentration.

[0057] As can be seen from Table 1, the correlation coefficient R of the standard curve of each fatty amine 2 > 0.9992, indicating that the analyte has a good linear relationship. The LODs and LOQs are in the ranges of 1.59 - 4.29 μg / Kg and 4.27 - 14.63 μg / Kg respectively. The intra-day and inter-day precisions of the analyte are in the ranges of 0.63 - 2.14% and 2.89 - 6.94% respectively. That is, this analytical method has a good linear relationship, high precision, low detection limit, and low quantification limit.

[0058] The reaction route diagram of the labeling reagent of the present invention with fatty amine is shown in Figure 5 ; The chromatographic separation diagram of 10 kinds of mixed fatty amine standards after labeling and derivatization is shown in Figure 6 . As can be seen from Figure 6 , the labeling reagent of the present invention can label fatty amines quickly, accurately, and sensitively, and is especially suitable for the separation and detection of trace and ultra-trace small molecule amine compounds in complex systems.

[0059] Example 3

[0060] A method for detecting the fatty amine content in fresh fish meat, the steps are as follows:

[0061] Take 1 g of fresh fish meat, cut it into pieces and grind it, place it in a 10 mL centrifuge tube, add 2 mL of water and 2 mL of acetonitrile, shake well and ultrasonicate for 15 min. Centrifuge to obtain the food supernatant, add 50 mg of Na 2 SO 4 , remove the residual moisture in the food to obtain the sample to be measured. Add 400 μL of boric acid-borax buffer solution, 20 μL of the above-mentioned sample to be measured, and 100 μL of acetonitrile solution of 4-(N-phenylphenanthroimidazolyl)-benzyl chloroformate into a 2 mL ampoule. After reacting at room temperature for 4 min, the derivatized solution is processed as follows:

[0062] (a) Add 50 μL of quenching agent acetic acid to stop the reaction, and let it stand for 5 - 10 minutes for injection analysis;

[0063] (b) Take 10 μL for high performance liquid chromatography-fluorescence analysis to obtain the chromatogram of fatty amines in the sample of fresh fish meat, as shown in Figure 7 . Substitute the peak areas of the obtained fatty amines into the corresponding working curves, and the concentrations of the fatty amines in the fresh fish meat can be obtained, and then the contents of the fatty amines can be obtained. Specifically, see Table 2.

[0064] Table 2

[0065]

[0066] As can be seen from Table 2, the fresh fish meat selected in this example only contains eight fatty amines: methylamine, ethylamine, propylamine, butylamine, pentylamine, hexylamine, heptylamine, and octylamine, and their concentrations are accurately measured.

[0067] Example 4

[0068] The recovery rate was evaluated by adding fatty amine standard solutions at three levels (5 mg / L, 10 mg / L, 15 mg / L) to the seafood product extract. The recovery rate formula is: (measured fatty amine concentration / theoretical fatty amine concentration) × 100%. Specifically:

[0069] (1) Obtain the sample to be measured of fresh fish meat according to Example 3, and detect the concentrations c1 of each fatty amine in the fresh fish meat;

[0070] (2) Add 400 μL of boric acid-borax buffer solution, 20 μL of the above-mentioned sample to be measured, and 100 μL of acetonitrile solution of 4-(N-phenylphenanthroimidazolyl)-benzyl chloroformate into a 2 mL ampoule in sequence. Then add mixed fatty amine standard samples with concentrations of 5 mg / L (the concentrations of C1 - C10 fatty amines are all 5 mg / L), 10 mg / L (the concentrations of C1 - C10 fatty amines are all 10 mg / L), and 15 mg / L (the concentrations of C1 - C10 fatty amines are all 15 mg / L) respectively. After reacting at room temperature for 4 min, the derivatized solution is processed as follows:

[0071] (a) Add 50 μL of quenching agent acetic acid to stop the reaction, and place it for 5 - 10 minutes for sample injection analysis;

[0072] (b) Take 10 μL for high performance liquid chromatography - fluorescence analysis to obtain a new chromatogram of fatty amines. Substitute the peak areas of each fatty amine obtained into the corresponding working curve, and the concentration c2 of each fatty amine can be obtained. Calculate the recovery rate according to the following formula, and the results are shown in Table 3.

[0073] Recovery rate = (c2 - c1) × 100% / concentration of mixed fatty amine standard sample.

[0074] Table 3

[0075]

[0076] As can be seen from Table 2, by adding fatty amine standard solutions at three levels (5 mg / L, 10 mg / L, 15 mg / L) to the seafood product extract to evaluate the recovery rate, the recovery rate is in the range of 88.5 - 100.8%. The recovery rate of the fatty amines of the present invention is good, indicating that the accuracy of this method is good.

[0077] In summary, the labeling reagent prepared by the present invention can label fatty amines quickly, accurately and sensitively. At the same time, the analysis method provided by the present invention has good linear relationship, high accuracy and precision, low detection line and quantification limit, and has broad application prospects.

[0078] The above is the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. Application of a phenanthroimidazole-based fluorescent amine compound labeling reagent, characterized in that, it is used for detecting the content of fatty amines in a sample, and the phenanthroimidazole-based fluorescent amine compound labeling reagent has the following chemical structural formula: 。 2. The application of the phenanthroimidazole-based fluorescent amine compound labeling reagent according to claim 1, characterized in that, the specific detection steps are as follows: (1) Solution preparation: Prepare a boric acid-borax buffer solution with a pH of 9.0; Prepare 5 portions of mixed fatty amine mixed standard solutions with concentrations of 10 nM, 100 nM, 1 μM, 10 μM, and 100 μM respectively; Prepare an acetonitrile solution of 4-(N-phenylphenanthroimidazolyl)-benzyl chloroformate with a concentration of 10 mM; (2) Working curve drawing: Sequentially add 400 μL of boric acid-borax buffer solution, 20 μL of mixed fatty amine standard solution, and 100 μL of acetonitrile solution of 4-(N-phenylphenanthroimidazolyl)-benzyl chloroformate into a 2 mL ampoule bottle. After reacting at room temperature for 4 minutes, the derivatized solution is processed as follows: (a) Add 50 μL of quenching agent acetic acid to stop the reaction, and let it stand for 5 - 10 minutes for injection analysis; (b) Take 10 μL of the reaction solution for high performance liquid chromatography-fluorescence analysis to obtain the chromatographic separation diagram of the mixed fatty amine standard. After derivatizing and performing high performance liquid chromatography-fluorescence analysis on the five portions of the mixed fatty amine standard in sequence, draw a working curve of fatty amine concentration with the fatty amine concentration as the abscissa and the peak area as the ordinate; (3) Fatty amine concentration detection: After derivatizing and labeling the sample to be tested with 4-(N-phenylphenanthroimidazolyl)-benzyl chloroformate according to the steps in (2), perform high performance liquid chromatography-fluorescence analysis. Substitute the obtained peak area into the working curve of fatty amine concentration to obtain the concentration of fatty amines in the sample.

3. The application of the phenanthroimidazole-based fluorescent amine compound labeling reagent according to claim 2, characterized in that, in the step (1), the fatty amine is a mixture of methylamine, ethylamine, propylamine, butylamine, pentylamine, hexylamine, heptylamine, octylamine, nonylamine, and decylamine.

4. The application of the phenanthroimidazole-based fluorescent amine compound labeling reagent according to claim 2, characterized in that, the steps for detecting the content of fatty amines in the sample of fresh raw fish are as follows: Take 1 g of fresh fish meat, cut it into pieces and grind it, place it in a 10 mL EP tube, add 2 mL of water and 2 mL of acetonitrile, shake well and sonicate for 15 min. Add 50 mg of Na 2 SO 4 , further remove the residual moisture in the food. After labeling according to the method in step (2), perform high performance liquid chromatography-fluorescence analysis. Substitute the obtained peak area into the working curve to obtain the concentration of each fatty amine in the fresh fish meat, and then obtain the content of each fatty amine.

5. The synthesis method of the phenanthroimidazole-based fluorescent amine compound labeling reagent described in claim 1, characterized in that, it includes: Step 1: Perform a substitution reaction and hydrolysis on phenanthroimidazole and p-hydroxymethylbenzaldehyde to obtain the intermediate 4-(N-phenylphenanthroimidazolyl)-benzyl alcohol; Step 2: React the intermediate 4-(N-phenylphenanthroimidazolyl)-benzyl alcohol with solid phosgene to obtain the target product 4-(N-phenylphenanthroimidazolyl)-benzyl chloroformate.

6. The synthesis method of the phenanthroimidazole-based fluorescent amine compound labeling reagent according to claim 5, characterized in that, the specific step 1 is: Dissolve phenanthraquinone, aniline, 4-(hydroxymethyl)benzaldehyde, and ammonium acetate in acetic acid. React under electromagnetic stirring in an oil bath at 105 °C for 4 h. After the reaction is completed, wait for the reaction solution to cool, then pour it into 500 mL of water and continuously stir to precipitate a light green solid. Filter by suction and wash twice with water. Air-dry the crude product. Dissolve the above product in 300 mL of 95% ethanol, add an aqueous potassium hydroxide solution, and react under electromagnetic stirring in an oil bath at 80 °C for 1 h. After the reaction is completed, evaporate to dryness using a rotary evaporator, and recrystallize twice with ethanol to obtain light green crystals.

7. The synthesis method of the phenanthroimidazole-based fluorescent amine-based compound labeling reagent according to claim 6, characterized in that, in step 1, the molar ratio of phenanthraquinone, aniline, 4-(hydroxymethyl)benzaldehyde, and ammonium acetate is 1:1.2:1:10, the reaction temperature is 105 °C, and the reaction time is 4 h.

8. The synthesis method of the phenanthroimidazole-based fluorescent amine-based compound labeling reagent according to claim 5, characterized in that, step 2 is specifically: Dissolve solid phosgene in dichloromethane. Under ice-bath cooling and stirring conditions, dropwise add triethylamine, and gradually dropwise add a dichloromethane solution containing the intermediate 4-(N-phenylphenanthroimidazolyl)-benzyl alcohol. React under ice bath for 3 h, and slowly raise the temperature to room temperature and react for 24 h. After the reaction is completed, filter by suction, and evaporate the filtrate to dryness under reduced pressure using a rotary evaporator, and recrystallize with dichloromethane to obtain the target product 4-(N-phenylphenanthroimidazolyl)-benzyl chloroformate.

9. The synthesis method of the phenanthroimidazole-based fluorescent amine-based compound labeling reagent according to claim 8, characterized in that, in step 2, 4-(N-phenylphenanthroimidazolyl)-benzyl chloroformate is recrystallized 2 times to obtain a white needle-like crystalline product; the molar ratio of 4-(N-phenylphenanthroimidazolyl)-benzyl alcohol to solid phosgene is 1:3, and the addition amount of triethylamine is 3% of the mass of solid phosgene.

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