A near-infrared fluorescent probe for detecting butyrylcholinesterase, and a preparation method and application thereof

Abstract

The present application relates to the field of fluorescence analysis detection technology, and more particularly to a near-infrared fluorescent probe for detection of butyrylcholinesterase, and a preparation method and application thereof. The fluorescent probe provided by the present application is based on a near-infrared fluorescent mother nucleus derived from julolidine, and realizes initial fluorescence quenching of the probe by introducing a p-hydroxybenzyl cyclopropanoyl ester recognition group. In the presence of BChE, the cyclopropanoyl ester group is specifically hydrolyzed by enzyme, triggering a self-destruction release process, and restoring the near-infrared emission of the fluorescent mother nucleus, so as to realize high-sensitivity and high-accuracy detection of BChE.

Description

Technical Field

[0001] This invention relates to the field of fluorescence analysis and detection technology, and in particular to a near-infrared fluorescent probe for the detection of butyrylcholinesterase, its preparation method, and its application. Background Technology

[0002] Butyrylcholinesterase (BChE) is an important serine hydrolase widely found in plasma, liver, nerve tissue, and various body fluids, primarily involved in the metabolism of choline esters. BChE activity levels are closely related to liver function impairment, acute inflammation, neurodegenerative diseases, pesticide poisoning, and various metabolic diseases. In particular, in serum samples, BChE is often used as an important biomarker for evaluating liver reserve function and disease progression.

[0003] Currently, the main methods for detecting BChE include colorimetric methods, electrochemical methods, enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC), and mass spectrometry. Although these methods have high accuracy, they generally suffer from problems such as complex sample pretreatment, long detection cycles, expensive equipment, and difficulty in achieving real-time dynamic monitoring, which limits their application in rapid clinical screening and point-of-care testing.

[0004] Fluorescent probe detection methods have gained widespread attention in enzyme activity analysis in recent years due to their advantages such as high sensitivity, rapid response, ease of operation, and the ability to achieve real-time dynamic imaging. However, most existing BChE fluorescent probes use short-wavelength fluorescent nuclei, which suffer from strong biological background fluorescence interference, poor tissue penetration, and limited detection sensitivity. Furthermore, some probes have insufficient selectivity of recognition groups, making them susceptible to interference from carboxylesterase (CE) and acetylcholinesterase (AChE), leading to decreased accuracy of detection results. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a near-infrared fluorescent probe, its preparation method and its application in the detection of butyrylcholinesterase. The probe provided by the present invention can achieve rapid, highly sensitive and highly accurate detection of BChE.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a near-infrared fluorescent probe having the structure shown in Formula 1: Formula 1.

[0007] The present invention provides a method for preparing the near-infrared fluorescent probe described above, comprising the following steps: under a protective atmosphere, mixing compound 3, a first acid-binding agent, an acyl transfer catalyst and compound 2, and carrying out a coupling reaction to obtain the near-infrared fluorescent probe; Compound 3; Compound 2.

[0008] Preferably, the molar ratio of compound 2 to compound 3 is 1:(1~1.5).

[0009] Preferably, the first acid-binding agent includes one or more of potassium carbonate, sodium carbonate, and cesium carbonate; the molar ratio of compound 3 to the first acid-binding agent is 1:(2~4).

[0010] Preferably, the acyl transfer catalyst is one or more of 4-dimethylaminopyridine, pyridine and imidazole; the molar ratio of compound 3 to the acyl transfer catalyst is 1:(0.5~1.5).

[0011] Preferably, the preparation method of compound 3 includes the following steps: adding p-hydroxybenzyl alcohol and cyclopropionyl chloride to dichloromethane, and carrying out an esterification reaction in the presence of a second acid-binding agent to obtain compound 3.

[0012] Preferably, the molar ratio of p-hydroxybenzyl alcohol to cyclopropionyl chloride is 1:1.5; and the temperature of the esterification reaction is 0~25℃.

[0013] This invention provides the application of the near-infrared fluorescent probe described above in the detection of butyrylcholinesterase for non-disease diagnostic and therapeutic purposes.

[0014] The present invention provides a kit for detecting butyrylcholinesterase, comprising the near-infrared fluorescent probe as described in claim 1, dimethyl sulfoxide, and a buffer solution.

[0015] Preferably, the buffer solution is PBS buffer with a concentration of 10 mM and a pH of 7.4.

[0016] This invention provides a near-infrared fluorescent probe. The fluorescent probe provided by this invention is based on a near-infrared fluorescent core derived from juloridine. By introducing a p-hydroxybenzylcyclopropionyl ester recognition group, the probe's initial fluorescence is quenched. In the presence of BChE, the cyclopropionyl ester group undergoes specific enzymatic hydrolysis, triggering a self-immolative elimination process, restoring the near-infrared emission of the fluorescent core, thereby achieving highly sensitive and accurate detection of BChE. Attached Figure Description

[0017] Figure 1 This is a standard curve of probe fluorescence intensity versus BChE concentration in Example 2. Detailed Implementation

[0018] This invention provides a near-infrared fluorescent probe having the structure shown in Formula 1: Formula 1; In this invention, the near-infrared fluorescent probe is denoted as Probe-BChE.

[0019] The near-infrared fluorescent probe provided by this invention uses a julloridin-type near-infrared fluorescent core as a chromophore and connects to a p-hydroxybenzylcycloalkylcarboxylic acid ester recognition unit via a carbamate bond.

[0020] The recognition group can undergo selective hydrolysis under the catalysis of butyrylcholinesterase, which further induces the self-destruction cleavage of the p-hydroxybenzyl group, releasing the free fluorescent nucleus and causing the probe to change from the fluorescence off (OFF) state to the fluorescence on (ON) state, thus achieving highly selective detection of BChE activity.

[0021] Based on the above principles, the near-infrared fluorescent probe of the present invention can be used for qualitative detection of BChE in serum samples; quantitative detection of BChE in serum samples; auxiliary diagnosis of liver function impairment-related diseases; rapid screening of clinical biological samples; and development of point-of-care testing (POCT) systems.

[0022] The present invention provides a method for preparing the near-infrared fluorescent probe described above, comprising the following steps: under a protective atmosphere, mixing compound 3, a first acid-binding agent, an acyl transfer catalyst and compound 2, and carrying out a coupling reaction to obtain the near-infrared fluorescent probe; Compound 3; Compound 2.

[0023] Unless otherwise specified, all raw materials used in this invention are commercially available products well known in the art.

[0024] In this invention, the preparation method of compound 3 preferably includes the following steps: adding p-hydroxybenzyl alcohol and cyclopropionyl chloride to dichloromethane, and carrying out an esterification reaction in the presence of a second acid-binding agent to obtain compound 3.

[0025] In this invention, the molar ratio of p-hydroxybenzyl alcohol to cyclopropionyl chloride is preferably 1:1.5. In this invention, the amount of dichloromethane used is not particularly limited, as long as it is sufficient to dissolve both p-hydroxybenzyl alcohol and cyclopropionyl chloride. In this invention, the second acid-binding agent is preferably triethylamine; the amount of triethylamine used is not particularly limited, and a well-known amount, generally 10-50 μL, is acceptable. In this invention, the esterification reaction temperature is preferably 0-25°C, and the reaction time is preferably 4-12 h.

[0026] After the esterification reaction is completed, the solvent is preferably removed from the obtained system under reduced pressure. The residue is purified by silica gel column chromatography using petroleum ether / ethyl acetate (v / v = 20:1) as the eluent to obtain compound 3.

[0027] In this invention, the preparation method of compound 2 preferably includes the following steps: Under an inert atmosphere, compound 1 and sodium carbonate were dissolved in water, and then dichloromethane was added to form a biphase reaction system. Subsequently, an aqueous solution of sodium dithionite was added dropwise, and the reaction was stirred at 40°C. After the reaction was completed, triethylamine was added dropwise to the system, and stirring was continued. The system was then cooled to 0°C, and a dichloromethane solution of triphosgene was added dropwise. The reaction was continued under an inert atmosphere to obtain compound 2.

[0028] Compound 1.

[0029] In this invention, compound 1 can be a commercially available product or prepared using methods well known in the art. The preparation method of compound 1 in this invention includes the following steps: dissolving 8-hydroxyjuloridin and N,N-diethyl-3-methoxy-4-nitrosoaniline in an isopropanol / water mixed solvent, heating to reflux, slowly adding perchloric acid to catalyze a condensation reaction, and continuously stirring the reaction overnight to obtain compound 1.

[0030] In this invention, the volume ratio of isopropanol to water is preferably 9:1; the molar ratio of 8-hydroxyjuloridine to N,N-diethyl-3-methoxy-4-nitrosoaniline is preferably 1:1; the amount of perchloric acid added is not particularly limited in this invention, and a catalyst amount of perchloric acid well known in the art can be used. In this invention, the condensation reaction time is preferably 12-24 h.

[0031] After the condensation reaction is completed, the present invention preferably removes the solvent from the obtained reaction system under reduced pressure, and the resulting residue is purified by silica gel column chromatography using dichloromethane / methanol (v / v = 10:1) as the eluent to obtain a dark blue solid compound 1.

[0032] Let's return to the preparation method of compound 2.

[0033] In this invention, under an inert atmosphere, compound 1 and sodium carbonate are dissolved in water, and then dichloromethane is added to form a two-phase reaction system. Subsequently, an aqueous solution of sodium dithionite is added dropwise, and the reaction is stirred at 40°C.

[0034] In this invention, the molar ratio of compound 1 to sodium carbonate is preferably 1:4; the amount of water used is not particularly limited, as long as it is sufficient to completely dissolve compound 1 and sodium carbonate. In this invention, the volume ratio of dichloromethane to water is preferably 6:7; the molar ratio of compound 1 to sodium dithionite is preferably 1:4; the concentration of the sodium dithionite aqueous solution is preferably 0.2 mmol / mL. The stirring reaction time at 40°C is preferably 1 h.

[0035] After the reaction was completed, triethylamine was added dropwise to the system and stirring was continued; then the system was cooled to 0°C, and a triphosgene solution in dichloromethane was added dropwise, and the reaction was continued under an inert atmosphere to obtain compound 2.

[0036] In this invention, the molar ratio of compound 1 to triethylamine is preferably 1:1.4; the molar ratio of compound 1 to triphosgene is preferably 1:(0.5~0.8), and in a specific embodiment, it is 1:0.6; the concentration of the triphosgene dichloromethane solution is preferably 0.06 mmol / mL. In this invention, the reaction is preferably continued for 1 h under an inert atmosphere.

[0037] After the reaction is complete, the present invention preferably uses dichloromethane for multiple extractions, combines the organic phases to obtain compound 2, which is directly used for the subsequent synthesis of compound 3 without further purification.

[0038] After obtaining compound 3 and compound 2, the present invention mixes compound 3, the first acid-binding agent, the acyl transfer catalyst and compound 2 under a protective atmosphere and carries out a coupling reaction to obtain the near-infrared fluorescent probe.

[0039] In this invention, the protective atmosphere is preferably a nitrogen atmosphere or an argon atmosphere.

[0040] In this invention, the molar ratio of compound 2 to compound 3 is preferably 1:(1~1.5), and in specific embodiments it can be 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4 or 1:1.5.

[0041] In this invention, the first acid-binding agent preferably includes one or more of potassium carbonate, sodium carbonate, and cesium carbonate; the molar ratio of the compound 3 to the first acid-binding agent is preferably 1:(2~4), and in specific embodiments it can be 1:2, 1:2.5, 1:3, 1:3.5 or 1:4.

[0042] In this invention, the acyl transfer catalyst is preferably one or more of 4-dimethylaminopyridine, pyridine, and imidazole; the molar ratio of compound 3 to the acyl transfer catalyst is preferably 1:(0.5~1.5), and in specific embodiments it can be 1:0.5, 1:0.8, 1:1, 1:1.2, or 1:1.5.

[0043] In this invention, the temperature of the coupling reaction is preferably 45°C, and the time of the coupling reaction is preferably 12~24h.

[0044] After completing the coupling reaction, the present invention preferably further includes post-treatment of the obtained reaction solution. The post-treatment preferably includes the following steps: washing the reaction solution three times with 1 M hydrochloric acid to remove inorganic salts and alkaline impurities, removing organic solvents under reduced pressure, purifying the obtained residue by silica gel column chromatography, using dichloromethane / methanol (v / v = 20:1) as the eluent to obtain the near-infrared fluorescent probe.

[0045] This invention provides the application of the near-infrared fluorescent probe described above in the detection of butyrylcholinesterase for non-disease diagnostic and therapeutic purposes.

[0046] In this invention, the detection can be qualitative or quantitative.

[0047] The detection is applicable to biological samples; the biological samples include: human serum, animal serum, plasma samples, liver tissue homogenate, cell lysate or clinical body fluid samples, preferably human serum samples.

[0048] The near-infrared fluorescent probe provided by this invention can be used for: auxiliary diagnosis of liver function damage, auxiliary analysis of inflammation-related diseases, rapid screening of clinical biological samples, development of point-of-care testing (POCT) systems, and fluorescence imaging analysis by detecting BChE; it is especially suitable for the rapid detection of BChE activity in serum samples.

[0049] The present invention provides a kit for detecting butyrylcholinesterase, comprising the above-mentioned near-infrared fluorescent probe, dimethyl sulfoxide and buffer solution.

[0050] In this invention, the buffer solution is preferably PBS buffer, preferably with a concentration of 10 mM and a pH of 7.4.

[0051] This invention also provides a method for using the above-mentioned kit: dissolve the near-infrared fluorescent probe in dimethyl sulfoxide to obtain a fluorescent probe solution; mix the fluorescent probe solution with the sample to be tested and a buffer solution to obtain a test solution; incubate the test solution at 37°C for 30 min, and then perform fluorescence emission spectroscopy detection with an excitation wavelength of 600~700 nm, and record the fluorescence emission spectrum in the range of 700~800 nm.

[0052] In this invention, the final concentration of the near-infrared fluorescent probe in the test solution is preferably 5 μM. The volume ratio of the fluorescent probe solution to the test sample is preferably 1:1; the volume ratio of the fluorescent probe solution to the buffer solution is preferably 1:98.

[0053] In a specific embodiment, the excitation wavelength can be 600, 620, 650, 680 or 700 nm; the present invention preferably records the emission spectrum in the range of 680~850 nm.

[0054] In this invention, the conditions for fluorescence emission spectroscopy detection further include: The excitation slit width is preferably 5~10 nm; in specific embodiments, it can be 5, 6, 7, 8, 9 or 10 nm. The preferred width of the emission slit is 5~10 nm; in specific embodiments, it can be 5, 6, 7, 8, 9 or 10 nm. The operating voltage of the photomultiplier tube is preferably 300~700 V, and in specific embodiments it can be 300, 400, 500, 600 or 700 V.

[0055] The kit provided by this invention can realize the qualitative or quantitative detection of BChE. When used for quantitative detection, this invention preferably adopts the standard curve method, plotting a standard curve with BChE concentration as the abscissa and fluorescence intensity as the ordinate, and obtaining the BChE concentration in the test solution according to the standard curve, thereby obtaining the BChE concentration in the test sample.

[0056] The near-infrared fluorescent probe, its preparation method, and its application in butyrylcholinesterase detection provided by the present invention are described in detail below with reference to the embodiments. However, these should not be construed as limiting the scope of protection of the present invention.

[0057] The synthesis routes of the probes in the following examples and comparative examples are shown in Equation 3: Formula 3.

[0058] When n=1, the Probe-BChE probe of Example 1 is obtained; when n=2, the probe of Comparative Example 1 is obtained; when n=3, the probe of Comparative Example 2 is obtained.

[0059] Example 1 8-Hydroxyjuloridin (757 mg, 4 mmol) was dissolved in an isopropanol / water mixture (v / v = 9:1, 20 mL) and heated to reflux. After the system stabilized, N,N-diethyl-3-methoxy-4-nitrosoaniline (833 mg, 4 mmol) and perchloric acid (70%, 300 μL) were slowly added, and the reaction was continued with stirring overnight. After the reaction was completed, the solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography using dichloromethane / methanol (v / v = 10:1) as the eluent to give compound 1 (418 mg, 30% yield), a deep blue solid.

[0060] Under nitrogen protection, compound 1 (0.5 mmol) and sodium carbonate (212 mg, 2 mmol) were dissolved in 7 mL of water, and then 6 mL of dichloromethane was added to form a biphase system. Sodium dithionite (400 mg, 2 mmol) was then dissolved in 10 mL of water and slowly added dropwise to the reaction system, and the mixture was stirred at 40 °C for 1 hour. Triethylamine (100 μL, 0.7 mmol) was then slowly added dropwise, and the reaction continued. The system was cooled to 0 °C, and triphosgene (100 mg, 0.3 mmol) dissolved in 5 mL of dichloromethane was slowly added dropwise, and the mixture was stirred again under nitrogen protection for 1 hour. After the reaction was complete, the mixture was extracted with dichloromethane (10 mL × 3), and the organic phases were combined to obtain compound 2, which was used directly in the next reaction.

[0061] p-Hydroxybenzyl alcohol and cyclopropionyl chloride were added to 10 mL of dichloromethane at a molar ratio of 1:1.5, and esterification was carried out in the presence of triethylamine. After the reaction was completed, the mixture was purified by silica gel column chromatography using petroleum ether / ethyl acetate (v / v = 20:1) as the eluent to give compound 3.

[0062] Under nitrogen protection, compound 3 (0.5 mmol), potassium carbonate (207 mg, 1.5 mmol), and 4-dimethylaminopyridine (61 mg, 0.5 mmol) were added to the reaction solution of compound 2, and the mixture was stirred overnight at 45 °C. After the reaction was complete, the reaction solution was washed three times with 1 M hydrochloric acid, the solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography using dichloromethane / methanol (v / v = 20:1) as the eluent to finally obtain the light blue solid target product Probe-BChE.

[0063] 1 H NMR (400 MHz, DMSO- d 6 ) δ 7.74 (d, J = 8.5 Hz, 1H), 7.25–7.35 (m,4H), 7.09 (d, J = 2.3 Hz, 1H), 7.02 (s, 1H), 6.95 (dd, J = 8.5, 2.3 Hz, 1H), 5.09 (s, 2H), 3.63 (m, 2H), 3.18–3.34 (m, 6H), 2.56–2.64 (m, 4H), 1.82–1.94(m, 4H), 1.52 (m, 1H), 1.04 (t, J = 7.1 Hz, 6H), 0.78–0.89 (m, 4H).

[0064] 13C NMR (100 MHz, DMSO- d 6 ) δ 170.1, 156.0, 154.9, 154.4, 150.6, 149.4,146.7, 141.6, 140.0, 132.4, 129.6, 126.6, 123.8, 120.5, 120.4, 120.3, 117.1,116.0, 98.2, 67.4, 50.2, 49.7, 44.6, 27.7, 26.7, 21.6, 21.1, 12.8, 12.5, 8.3.

[0065] ESI-MS: C 34 H 38 N3O5 + Calculated value: 568.2806; Actual measurement: 568.2801. The above results confirm that the obtained product is the target product.

[0066] Example 2 Probe-BChE is used for BChE standard testing: Experimental group: Probe-BChE solid was dissolved in DMSO to prepare a probe stock solution with a final concentration of 1 mM, which was stored at -20℃ protected from light. 10 μL of the probe stock solution was added to PBS buffer (10 mM, pH = 7.4), followed by different volumes of BChE standard solution. The volume was then adjusted to 1000 μL using PBS buffer to obtain the test solution. The final BChE concentrations in the test solutions were 0 U / L, 20 U / L, 50 U / L, 100 U / L, 200 U / L, 400 U / L, 600 U / L, and 800 U / L, respectively; the final probe concentration was 5 μM. After incubating the test solutions at 37℃ for 30 min, the results were detected using a fluorescence spectrophotometer.

[0067] The detection conditions were as follows: excitation wavelength set to 650 nm; excitation and emission slit widths of 5 nm and 10 nm, respectively; photomultiplier tube operating voltage of 700 V; and emission spectra recorded in the range of 680–850 nm. A standard curve was plotted with BChE concentration on the x-axis and fluorescence intensity at the fluorescence emission peak on the y-axis.

[0068] The results showed that, within the range of 0–800 U / L, the probe fluorescence intensity exhibited a good linear relationship with the BChE concentration: F = 18.72C + 1256 (R0). 2 =0.9978), such as Figure 1As shown. According to the 3σ / k method, the near-infrared fluorescent probe provided by this invention has a high detection sensitivity of 0.3 U / L for BChE, which can meet the needs of rapid analysis of trace BChE in serum samples.

[0069] Control group: The only difference from the experimental group was that the BChE standard solution was replaced with PBS buffer.

[0070] The results showed that after incubation at 37℃ for 30 min, the fluorescence signal of Probe-BChE remained basically stable in the control group, with only weak background fluorescence observed at 740 nm and no significant increase in fluorescence intensity.

[0071] Example 3 Detection of Probe-BChE in human serum samples: Serum samples from healthy volunteers were collected, centrifuged, and then used for later use. Known concentrations of BChE standard solution were added to the serum samples for spiked recovery experiments at concentrations of 100 U / L, 300 U / L, and 500 U / L. 10 μL of probe stock solution was added to PBS buffer, followed by 10 μL of serum sample. The PBS buffer was then brought to a final volume of 1000 μL, and fluorescence detection was performed according to the detection conditions described in Example 2. A standard clinical colorimetric method was used as a control method to simultaneously determine the BChE concentration in the serum samples.

[0072] Table 1. Analytical results of BChE concentration in serum samples (n = 5)

[0073] As shown in Table 1, the near-infrared fluorescent probe Probe-BChE provided by this invention can achieve rapid detection of butyrylcholinesterase in serum samples, and the detection results are basically consistent with those of conventional clinical colorimetric methods. The spiked recoveries obtained by the fluorescence method are 97.0%~101.6%, and the relative standard deviations (RSDs) are all less than 5%, indicating that the method has good accuracy, stability and repeatability, and can meet the needs of rapid analysis and detection of BChE in serum samples, thus having good clinical application value.

[0074] Comparative Example 1 The only difference from Example 1 is that cyclopropionyl chloride is replaced with cyclobutyryl chloride; all other preparation conditions are the same as in Example 1. The probe obtained differs from the probe of Example 1 in that the recognition group is replaced with a cyclobutyryl ester group instead of a cyclopropionyl ester group.

[0075] The BChE response performance of the probe was evaluated using the same method as in Example 2.

[0076] The results showed that, within the range of 0–800 U / L, the probe prepared in Comparative Example 1 only exhibited weak fluorescence enhancement after the addition of BChE, and the fluorescence intensity did not show a good linear relationship with the BChE concentration (R0). 2 <0.90), which is insufficient to meet the sensitivity requirements for detecting BChE in serum samples.

[0077] The reason is speculated to be that the large steric hindrance of the cyclobutyryl ester group reduces the recognition and catalytic efficiency of BChE for the substrate, thus making it difficult for the probe to undergo effective enzymatic hydrolysis and subsequent self-destruction release process.

[0078] Therefore, the probe prepared in Comparative Example 1 is not suitable as a BChE fluorescence detection probe.

[0079] Comparative Example 2 The only difference from Example 1 is that cyclopropionyl chloride is replaced with cyclopentanoyl chloride; all other preparation conditions are the same as in Example 1. The probe obtained differs from the probe of Example 1 in that the recognition group is replaced with "cyclopentanoyl ester group".

[0080] The BChE response performance of the probe was evaluated using the same method as in Example 2.

[0081] The results showed that, under the same experimental conditions, the probe prepared in Comparative Example 2 showed almost no significant fluorescence enhancement after the addition of BChE, and no reliable linear relationship between concentration and fluorescence intensity was established, making it impossible to accurately detect BChE in serum samples.

[0082] The reason is speculated to be that the cyclopentanoyl ester group has greater steric hindrance, which reduces the matching between the probe and the BChE active pocket, significantly reduces the efficiency of enzymatic hydrolysis, and thus cannot effectively trigger the fluorescence recovery of the probe.

[0083] Therefore, the probe prepared in Comparative Example 2 is not suitable as a BChE fluorescence detection probe.

[0084] The experimental results of Comparative Examples 1 and 2 show that the ring size of the recognition group has a significant impact on the BChE recognition performance of the probe. Compared with cyclobutyryl ester and cyclovaleryl ester structures, the cyclopropionyl ester structure has better spatial matching and catalytic adaptability to the BChE active site, enabling efficient enzymatic hydrolysis and significant fluorescence recovery. Therefore, cyclopropionyl ester is the preferred recognition group of this invention.

[0085] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A near-infrared fluorescent probe, characterized in that, It has the structure shown in Equation 1: Formula 1.

2. The method for preparing the near-infrared fluorescent probe according to claim 1, characterized in that, Includes the following steps: Under a protective atmosphere, compound 3, the first acid-binding agent, the acyl transfer catalyst and compound 2 were mixed and coupled to obtain the near-infrared fluorescent probe. Compound 3; Compound 2.

3. The preparation method according to claim 2, characterized in that, The molar ratio of compound 2 to compound 3 is 1:(1~1.5).

4. The preparation method according to claim 2, characterized in that, The first acid-binding agent includes one or more of potassium carbonate, sodium carbonate, and cesium carbonate; the molar ratio of compound 3 to the first acid-binding agent is 1:(2~4).

5. The preparation method according to claim 2, characterized in that, The acyl transfer catalyst is one or more of 4-dimethylaminopyridine, pyridine, and imidazole; the molar ratio of compound 3 to the acyl transfer catalyst is 1:(0.5~1.5).

6. The preparation method according to claim 2, characterized in that, The preparation method of compound 3 includes the following steps: adding p-hydroxybenzyl alcohol and cyclopropionyl chloride into dichloromethane, and carrying out an esterification reaction in the presence of a second acid-binding agent to obtain compound 3.

7. The preparation method according to claim 6, characterized in that, The molar ratio of p-hydroxybenzyl alcohol to cyclopropionyl chloride is 1:1.5; the temperature of the esterification reaction is 0~25℃.

8. The use of the near-infrared fluorescent probe of claim 1 in the detection of butyrylcholinesterase for non-disease diagnostic and therapeutic purposes.

9. A kit for detecting butyrylcholinesterase, characterized in that, It includes the near-infrared fluorescent probe, dimethyl sulfoxide, and buffer solution as described in claim 1.

10. The reagent kit according to claim 9, characterized in that, The buffer solution is PBS buffer with a concentration of 10 mM and a pH of 7.4.

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