A Golgi apparatus-targeted near-infrared fluorescent probe for detecting chymotrypsin, and its preparation method and application

Abstract

The present invention provides a Golgi-targeted near-infrared fluorescent probe for detecting chymotrypsin, as well as its preparation method and application. The structural formula is as follows: #imgabs0#, wherein R is selected from #imgabs1#. The probe of the present invention uses a hemicyanine derivative as a parent nucleus, introduces p-aminobenzenesulfonamide as a Golgi-targeting group, and uses 4-bromobutyrate or 3-phenylpropionate as a recognition group for detecting chymotrypsin. Chymotrypsin-induced ester group cleavage is accompanied by a significant enhancement of the fluorescent signal. The probe can effectively detect chymotrypsin from different proteases, amino acids, and yin and yang examples, with good selectivity and high sensitivity. The probe is accompanied by a distinct color change visible to the naked eye when acting on chymotrypsin, and exhibits good Golgi targeting capability, resulting in successful applications for chymotrypsin detection and imaging in biological systems.

Description

Technical Field

[0001] The invention belongs to the technical field of organic synthesis, and particularly relates to a Golgi apparatus-targeted near-infrared fluorescent probe for detecting chymotrypsin, and a preparation method and application thereof. Background Art

[0002] Chymotrypsin (Chy), as a serine protease, plays an important role in maintaining physiological functions such as protein digestion, immune response and tissue repair.

[0003] Traditional methods for detecting trace molecules, such as mass spectrometry, high-performance liquid chromatography, Western blotting, and enzyme-linked immunosorbent assay (ELISA), often require specialized equipment and suffer from insufficient sensitivity, high costs, time-consuming procedures, and cumbersome procedures. Fluorescent probe imaging technology, with its advantages of fast response, high sensitivity, ease of use, non-invasiveness, and good biocompatibility, has rapidly developed. Using techniques such as confocal microscopy and fluorescence imaging, researchers have studied biomolecules in organelles such as mitochondria, lysosomes, and the endoplasmic reticulum. However, probes for in situ investigation of small molecules within the Golgi apparatus are currently relatively rare. In recent years, numerous serine proteases within the Golgi apparatus have been reported to be associated with disease targets, and aberrant expression of Golgi proteases has become a research hotspot. Fluorescent sensors for serine proteases such as furin and elastase have been reported. However, Golgi-targeted fluorescent probes are still in their infancy. While various targeting groups have been reported, the localization mechanisms of most remain unclear, further hindering their development.

[0004] Currently, most fluorescent probes for chymotrypsin detection are mitochondrial-targeted, while near-infrared fluorescent probes targeting the Golgi apparatus for chymotrypsin detection have not been reported. Whether the Golgi apparatus and chymotrypsin are linked during disease progression warrants further exploration. Fluorescent probes have become powerful tools for observing small molecules within cells and are of great value in biomedical research. Applying fluorescent probe technology to the study of chymotrypsin and the Golgi apparatus could provide new possibilities and directions for drug development and cancer treatment. Therefore, developing a reliable and sensitive Golgi-targeted chymotrypsin detection method is of great significance for drug development and clinical diagnosis. Summary of the Invention

[0005] To overcome the problems of the prior art, the present invention provides a Golgi-targeted fluorescent probe for detecting chymotrypsin. This fluorescent probe responds rapidly to chymotrypsin, exhibits good selectivity and linearity, and has been successfully used for chymotrypsin imaging in the Golgi apparatus of living cells and in zebrafish.

[0006] The present invention also provides a preparation method and application of the fluorescent probe.

[0007] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] A Golgi apparatus-targeted fluorescent probe for detecting chymotrypsin, with the structural formula as follows:

[0009] Where R is selected from That is, the fluorescent probe is selected from the following compounds:

[0010]

[0011] The method for preparing the above-mentioned fluorescent probe targeting Golgi apparatus for detecting chymotrypsin comprises the following steps:

[0012] (1) Cyclohexanone, PBr3 and DMF react in dichloromethane to obtain compound I;

[0013] (2) 2,4-dihydroxybenzaldehyde reacts with 3,4-dihydro-2H-pyran in the presence of pyridinium p-toluenesulfonate as a catalyst to obtain compound II;

[0014] (3) Compound I, Compound II and potassium carbonate react in DMF to obtain Compound III;

[0015] (4) Compound III reacts with trifluoroacetic acid in dichloromethane with stirring to obtain compound IV;

[0016] (5) reacting p-aminobenzenesulfonamide with chloroacetyl chloride to obtain compound V;

[0017] (6) Compound V reacts with 2,3,3-trimethyl-3H-indole to obtain compound VI;

[0018] (7) Compound IV, 4-bromobutyryl chloride and triethylamine react in dichloromethane to obtain compound VII

[0019] (8) Compound IV, 3-phenylpropionyl chloride and triethylamine react in dichloromethane to obtain compound VIII

[0020] (9) Compound VI reacts with compound VII in ethanol to obtain the fluorescent probe CYB-C1;

[0021] (10) Compound VI reacts with compound VIII in ethanol to obtain the fluorescent probe CYB-C2.

[0022] Preferably, the preparation process of compound I in step (1) is as follows: under ice bath conditions, PBr3 is dropped into a mixed solution of DMF and dichloromethane, stirred for 0.5h to 1h, and then cyclohexanone is dropped into the above solution, stirred and reacted until complete, and then neutralized to neutrality, the product is extracted with dichloromethane, and concentrated by rotary evaporation to obtain the target product.

[0023] Preferably, the preparation process of compound II in step (2) is as follows: 2,4-dihydroxybenzaldehyde and pyridinium p-toluenesulfonate are weighed separately and added to dichloromethane, and then 3,4-dihydro-2H-pyran is added. After stirring and refluxing, the reaction is completed, followed by rotary evaporation and column chromatography to obtain compound II.

[0024] Preferably, the preparation process of compound III in step (3) is as follows: adding the prepared compound I, compound II and potassium carbonate to DMF, stirring at 35-45° C. until the reaction is complete, cooling to room temperature, filtering and concentrating to obtain a concentrated solution of compound III.

[0025] Preferably, the preparation process of compound IV in step (4) is as follows: dissolving the concentrated solution obtained in step (3) in dichloromethane, adding trifluoroacetic acid to adjust the pH to 6-7, stirring at 35-45°C for complete reaction, cooling to room temperature, filtering, washing with ethanol, and drying to obtain compound IV.

[0026] Preferably, the preparation process of compound V in step (5) is as follows: p-aminobenzenesulfonamide is weighed and added to acetone, chloroacetyl chloride is added dropwise, and after stirring at 70-80° C. for complete reaction, the mixture is cooled to room temperature, ice water is added, and the obtained mixture is stirred and filtered. The collected solid is washed with ice water, and then recrystallized with ethanol, filtered, and the obtained solid is dried to obtain compound V.

[0027] Preferably, the preparation process of compound VI in step (6) is as follows: under a nitrogen atmosphere, the compound V obtained in step (5) is dissolved in acetonitrile, 2,3,3-trimethyl-3H-indole is added, and the reaction is refluxed until completion, the reaction solution is vacuum evaporated, dichloromethane is added, and the reaction solution is filtered under reduced pressure to obtain a solid compound VI.

[0028] Preferably, the preparation process of compound VII in step (7) is as follows: under a nitrogen atmosphere, compound IV and triethylamine are added to dichloromethane, 4-bromobutyryl chloride is added under ice bath conditions, and after stirring for complete reaction, the mixture is rotary evaporated and column chromatography is performed to obtain compound VII.

[0029] Preferably, the preparation process of compound VII in step (8) is as follows: under a nitrogen atmosphere, compound IV and triethylamine are added to dichloromethane, 3-phenylpropionyl chloride is added under ice bath conditions, and after stirring for complete reaction, the mixture is rotary evaporated and column chromatography is performed to obtain compound VII.

[0030] Preferably, the preparation process of the fluorescent probe CYB-C1 in step (9) is as follows: under a nitrogen atmosphere, compound VII and compound VI are dissolved in ethanol, stirred and refluxed until a solid precipitate is formed, filtered, and the obtained solid is washed with ethyl acetate and then subjected to column chromatography to obtain the probe CYB-C1.

[0031] Preferably, the preparation process of the fluorescent probe CYB-C2 in step (10) is as follows: under a nitrogen atmosphere, compound VIII and compound VI are dissolved in ethanol, stirred and refluxed until a solid precipitate is formed, filtered, and the obtained solid is washed with ethyl acetate and then column chromatography to obtain the probe CYB-C2.

[0032] Preferably, in step (1), the molar ratio of PBr3, DMF and cyclohexanone is (2.5-3.0):(2.0-2.5):1.

[0033] Preferably, in step (2), the molar ratio of 2,4-dihydroxybenzaldehyde, 3,4-dihydro-2H-pyran and pyridinium p-toluenesulfonate is 1:(2.0-2.5):0.05.

[0034] Preferably, in step (3), the molar ratio of compound I, compound II and potassium carbonate is 1:1:3.

[0035] Preferably, in step (5), the molar ratio of p-aminobenzenesulfonamide to chloroacetyl chloride is 1:(1-1.5).

[0036] Preferably, in step (6), the molar ratio of compound V to 2,3,3-trimethyl-3H-indole is 1:(1-1.5).

[0037] Preferably, in step (7), the molar ratio of compound IV, triethylamine and 4-bromobutyryl chloride is 1:(1-2):(1-2).

[0038] Preferably, in step (8), the molar ratio of compound IV, triethylamine and 3-phenylpropionyl chloride is 1:(1-2):(1-2).

[0039] Preferably, in step (9), the molar ratio of compound VII to compound VI is 1:(1-1.5).

[0040] Preferably, in step (10), the molar ratio of compound VII to compound VI is 1:(1-1.5).

[0041] The fluorescent probe is used in the detection of chymotrypsin for the purpose of non-disease diagnosis.

[0042] Application of the above fluorescent probes in the preparation of cell fluorescence imaging agents.

[0043] The application of the fluorescent probe in the preparation of a Golgi apparatus-targeted fluorescent probe.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] 1. The ester bond of the fluorescent probe of this invention can be hydrolyzed and cleaved by chymotrypsin, accompanied by a significant increase in NIR fluorescence emission. The probe can also rapidly distinguish chymotrypsin from other proteases, amino acids, or bioactive small molecules such as anions and cations based on their different kinetic rates. Furthermore, the probe exhibits a significant change in fluorescence intensity accompanied by a distinct color change, visible to the naked eye.

[0046] 2. The fluorescent probe of this invention features simple synthesis, a high molar absorption coefficient in the NIR region, and excellent biocompatibility. The probe has been successfully used for fluorescence imaging in living cells and zebrafish, and its Golgi localization has been successfully verified. The probe can sensitively and selectively identify changes in chymotrypsin levels in cells and zebrafish, suggesting promising biomedical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 The synthetic route of the near-infrared fluorescent probe prepared in Example 1;

[0048] Figure 2 The H NMR spectrum of the fluorescent probe CYB-C1 prepared in Example 1;

[0049] Figure 3 The nuclear magnetic carbon spectrum of the fluorescent probe CYB-C1 prepared in Example 1;

[0050] Figure 4 High-resolution mass spectrometry of the fluorescent probe CYB-C1 prepared in Example 1;

[0051] Figure 5 The H NMR spectrum of the fluorescent probe CYB-C2 prepared in Example 1;

[0052] Figure 6 The nuclear magnetic carbon spectrum of the fluorescent probe CYB-C2 prepared in Example 1;

[0053] Figure 7 High-resolution mass spectrometry of the fluorescent probe CYB-C2 prepared in Example 1;

[0054] Figure 8 Spectral properties of the near-infrared fluorescent probe prepared in Example 1; a, b are respectively graphs showing the fluorescence spectrum change of the fluorescent probe CYB-C1 with different chymotrypsin concentrations, the corresponding relationship between the fluorescence intensity at 710 nm and the chymotrypsin concentration after response to different chymotrypsin concentrations, and the linear relationship between the fluorescence intensity at 710 nm; c, d are respectively graphs showing the fluorescence spectrum change of the fluorescent probe CYB-C2 with different chymotrypsin concentrations, the corresponding relationship between the fluorescence intensity at 710 nm and the chymotrypsin concentration after response to different chymotrypsin concentrations, and the linear relationship between the fluorescence intensity at 710 nm;

[0055] Figure 9Evaluation of the response speed of the near-infrared fluorescent probe prepared in Example 1 for identifying chymotrypsin; a is the corresponding relationship between the fluorescence intensity at 710 nm and time after the fluorescent probe CYB-C1 reacts with chymotrypsin; b is the corresponding relationship between the fluorescence intensity at 710 nm and time after the fluorescent probe CYB-C2 reacts with chymotrypsin;

[0056] Figure 10 The selectivity test of the near-infrared fluorescent probe prepared in Example 1 for identifying chymotrypsin; a is the selectivity test result of the fluorescent probe CYB-C1, and b is the selectivity test result of the fluorescent probe CYB-C2; in the figure, from left to right: (1) blank (2) Chy (3) AChE (4) BChE (5) trypsin (6) lysozyme (7) bovine serum albumin (8) lipase (9) pepsin (10) cellulase (11) Ser (12) Glu (13) Met (14) Phe (15) Cys (16) GSH (17) Hys (18) Tyr (19) Lys (20) Leu (21) Mg 2+ (22)Cu 2+ (23)Zn 2+ (24)Fe 2+ (25)Co 2+ (26)Gd 2+ (27)F - (28) NO2 - (29)ClO4 - (30)SCN - .

[0057] Figure 11 This is a biological imaging image of the near-infrared fluorescent probe CYB-C2 prepared in Example 1 when detecting chymotrypsin in living cells;

[0058] Figure 12 This is a biological imaging image of the localization ability test of the near-infrared fluorescent probe CYB-C2 prepared in Example 1 in living cells;

[0059] Figure 13 This is a biological imaging image of the near-infrared fluorescent probe CYB-C2 prepared in Example 1 detecting chymotrypsin in zebrafish. DETAILED DESCRIPTION

[0060] The present invention will be further described below with reference to the embodiments and drawings, but they are not intended to limit the present invention.

[0061] Example 1

[0062] The preparation process of the fluorescent probe in this embodiment specifically includes the following steps:

[0063] (1) Preparation of Compound Ⅰ

[0064] The synthetic route is as follows:

[0065]

[0066] Under ice-cooling conditions, PBr₃ (14.3 mL, 153 mmol) was slowly added dropwise to a mixture of DMF (10.5 mL, 137 mmol) and CH₂Cl₂ (60 mL) and stirred for 40 min. Cyclohexanone (5 mL, 56.5 mmol) was then slowly added. The ice-cooling bath was removed and the mixture was stirred at room temperature for 30 h. After the reaction, the reaction solution was slowly added to 100 mL of ice-cold water. The pH was adjusted to neutral with anhydrous sodium carbonate. The mixture was extracted with CH₂Cl₂ (3 × 50 mL). The organic phases were combined, washed with saturated brine, dried over Na₂SO₄, and distilled under reduced pressure to afford a reddish-brown oil. A total of 8.6 g of Compound I was obtained, with a yield of 81.1%, and the mixture was carried on to the next step without purification.

[0067] (2) Preparation of Compound II

[0068] The synthetic route is as follows:

[0069]

[0070] 2,4-Dihydroxybenzaldehyde (2.76 g, 20 mmol) and PPTS (pyridinium p-toluenesulfonate, 0.25 g, 1 mmol) were added to a 100 ml round-bottom flask containing 25 ml of CH2Cl2. Under nitrogen, 3,4-dihydro-2H-pyran (4.15 mL, 45.5 mmol) was added to the reaction flask and refluxed at 40°C. After completion of the reaction, the mixture was concentrated and purified by silica gel column chromatography to yield approximately 3.90 g of Compound II as a white solid in an approximately 88% yield. The H NMR spectrum of Compound II is as follows: 1 H NMR (400MHz, CDCl3) δ11.36(s,1H),9.72(s,1H),7.43(d,J=8.6Hz,1H),6.65(dd,J=8.6,2.3Hz,1H),6.62(d,J=2.2Hz,1H ),5.50(t,J=3.2Hz,1H),3.86–3.78(m,1H),3.66–3.59(m,1H),1.88–1.85(m,2H),1.72–1.66(m,2H),1.63–1.56(m,2H). 13 C NMR (100MHz, CDCl3) δ194.69,164.44,164.26,135.40,115.86,109.50,103.77,96.32,62.29,30.05,25.06,18.52.

[0071] (3) Preparation of Compound 3

[0072] The synthetic route is as follows:

[0073]

[0074] Under nitrogen, anhydrous potassium carbonate (3.73 g, 27 mmol), compound I (1.7 g, 9 mmol), and compound II (2.00 g, 9 mmol) were added to a round-bottom flask containing 10 mL of DMF and stirred at 40°C. The reaction solution was filtered under reduced pressure to remove the potassium carbonate solid, and then concentrated to obtain compound III, which was directly used in the next step without purification.

[0075] (4) Preparation of Compound IV

[0076] The synthetic route is as follows:

[0077]

[0078] 5 mL of CH2Cl2 and compound III were transferred into a 50 mL round-bottom flask. After adjusting the pH to 6 with TFA, the mixture was heated in a constant-temperature water bath at 40°C with stirring. After the reaction was complete, the mixture was filtered under reduced pressure and washed with icy ethanol to obtain about 1.22 g of compound IV as a yellow solid product with a yield of 59.4%. The H NMR spectrum of compound IV ( 1 H NMR) and carbon spectroscopy ( 13 C NMR) data are as follows: 1 H NMR (400MHz, DMSO-d6) δ10.17 (s, 1H), 7.17 (d, J = 8.2Hz, 1H), 6.90 (s, 1H), 6. 62–6.57(m,2H),2.54–2.50(m,2H),2.27(t,J=6.0Hz,2H),1.65–1.56(m,2H). 13 C NMR (100MHz, DMSO-d6) δ186.10,160.33,159.72,152.76,128.13,127.62,124.71,113.06,112.01,111.30,101.88,28.95,21.23,20.01.

[0079] (5) Preparation of Compound V

[0080] The synthetic route is as follows:

[0081]

[0082] Under nitrogen, p-aminobenzenesulfonamide (1.72 g, 10 mmol) was suspended in acetone. Chloroacetyl chloride (1.34 g, 12 mmol) was added dropwise at room temperature. The mixture was stirred at 70°C for 1 hour. After the reaction, the mixture was cooled to room temperature. Ice water was added and stirring continued for 10 minutes. The mixture was filtered under reduced pressure and washed with ice water. Multiple recrystallizations from ethanol afforded 2.36 g of a white solid in a 95.1% yield. The H NMR and C NMR data for Compound V are as follows: 1 H NMR (400MHz, DMSO-d6) δ10.62(s,1H),7.76(m,4H),7.28(s,2H),4.30(s,2H). 13 C NMR (100MHz, DMSO-d6) δ165.18,141.34,138.96,126.80,118.99,43.54.

[0083] (6) Preparation of Compound VI

[0084] The synthetic route is as follows:

[0085]

[0086] Under nitrogen protection, compound V (1.29 g, 5 mmol) was dissolved in CH3CN, 2,3,3-trimethyl-3H-indole (0.8 g, 6 mmol) was added, and the mixture was stirred under reflux at 85°C for 12 hours under nitrogen protection. After the reaction, the solvent was removed by vacuum rotary evaporation, CH2Cl2 solution was added, and the mixture was filtered under reduced pressure to obtain approximately 1.69 g of compound VI with a yield of 91.2%. The H NMR spectrum of compound VI ( 1 H NMR) and carbon spectroscopy ( 13 C NMR) data are as follows: 1 H NMR(400MHz,DMSO-d6)δ11.88(s,1H),7.98(s,1H),7.87(s,1H),7.84–7.7 5(m,4H),7.63(s,2H),7.32(s,2H),5.72(s,2H),2.87(s,3H),1.59(s,6H). 13 C NMR (100MHz, DMSO-d6) δ199.75,162.35,141.39,141.08,139.27,129.58,129.14,126.78,123.63,118.96,114.90,54.45,50.27,22.11,14.47.

[0087] (7) Preparation of Compound VII

[0088] The synthetic route is as follows:

[0089]

[0090] Under nitrogen, compound IV (228 mg, 0.1 mmol), triethylamine (202 mg, 0.2 mmol), and 5 mL of CHCl were mixed in an ice-water bath for 5 minutes. 4-Bromobutyryl chloride (183 mg, 0.1 mmol) was then added and stirred for 5 minutes. The ice bath was removed and the mixture was stirred at room temperature for 1 hour. After the reaction, the solvent was removed and the compound was purified by silica gel column chromatography with CHCl:CHOH (60:1) to afford VII (245.1 mg) of a yellow solid in a 65.2% yield.

[0091] (8) Preparation of Compound VIII

[0092] The synthetic route is as follows:

[0093]

[0094] Under nitrogen, compound IV (228 mg, 0.1 mmol), triethylamine (202 mg, 0.2 mmol), and 5 mL of CHCl were mixed in an ice-water bath for 5 minutes. 3-Phenylpropionyl chloride (168 mg, 0.1 mmol) was then added and stirred for 5 minutes. The ice bath was removed, and the mixture was stirred at room temperature for 1 hour. After the reaction, the solvent was removed and the compound was purified by silica gel column chromatography with CHCl:CHOH (60:1) to afford VII (265.0 mg) of a yellow solid in a 73.6% yield.

[0095] (9) Preparation of probe CYB-C1

[0096] The synthetic route is as follows:

[0097]

[0098] Under nitrogen protection, compound VI (37.2 mg, 0.01 mmol), compound VII (41.4 mg, 0.012 mmol), and 5 mL of ethanol were added to a 25 mL round-bottom flask and refluxed at 80°C for 12 h. After the reaction, the mixture was cooled to room temperature and the precipitated solid was filtered under reduced pressure. The resulting solid was purified by silica gel column chromatography with CH2Cl2:CH3OH (9:1) to obtain 41 mg of probe CYB-C1 with a yield of 55%. The H NMR spectrum of compound CYB-C1 ( 1 H NMR), carbon spectrum ( 13 C NMR) and high-resolution mass spectrometry such as Figures 2 to 5 The specific data are as follows: 1H NMR (400MHz, DMSO-d6) δ11.42 (s, 1H), 8.65 (d, J = 15.1Hz, 1H), 7.81 (m, 5H), 7.64 (t,J=7.3Hz,2H),7.58–7.47(m,4H),7.32(s,2H),7.18(d,J=8.5Hz,1H),6.71(d ,J=15.3Hz,1H),5.58(s,2H),3.73(dt,J=45.3,6.5Hz,2H),2.82(t,J=7.3Hz,2H ),2.73(t,J=5.9Hz,2H),2.63(d,J=6.1Hz,2H),2.26–2.09(m,2H),1.82(s,8H). 13 C NMR(100MHz,DMSO-d6)δ180.09,170.81,163.60,160.00,152.63,152.45,146 .08,141.80,141.75,141.08,139.15,131.89,129.78,128.99,128.41,127.44 ,126.86,122.87,119.61,119.54,118.93,114.81,112.91,109.97,106.03,50 .78,44.49,30.95,28.58,27.54,27.27,23.69,19.69,12.66.HR-MS(ESI):m / z calcd for C 37 H 37 BrN3O6S + :730.1581,found:730.1584.

[0099] (10) Preparation of probe CYB-C2

[0100] The synthetic route is as follows:

[0101]

[0102] Under nitrogen protection, compound VI (37.2 mg, 0.01 mmol), compound VIII (43.2 mg, 0.012 mmol), and 5 mL of ethanol were added to a 25 mL round-bottom flask and refluxed at 80°C for 12 h. After the reaction, the mixture was cooled to room temperature and the precipitated solid was filtered under reduced pressure. The resulting solid was purified by silica gel column chromatography with CH2Cl2:CH3OH (9:1) to obtain 42 mg of the probe CYB-C2 with a yield of 59%. The H NMR spectrum of CYB-C2 ( 1 H NMR), carbon spectrum ( 13 C NMR) and high-resolution mass spectrometry such as Figures 3 to 7 The specific data are as follows: 1 H NMR (400MHz, DMSO-d6) δ11.78 (s, 1H), 8.64 (d, J = 15.0Hz, 1H), 7.82 (m, 5H), 7. 68(d,J=7.9Hz,1H),7.61(d,J=8.5Hz,1H),7.57–7.47(m,3H),7.34(m,7H),7.2 7(m,1H),7.07(dd,J=8.4,2.2Hz,1H),6.74(d,J=15.0Hz,1H),5.63(s,2H),3. 01(t,J=4.1Hz,4H),2.72(t,J=6.0Hz,2H),2.63(d,J=6.2Hz,2H),1.82(s,8H). 13 C NMR (100MHz, DMSO-d6) δ180.05,170.81,163.71,159.86,152.60,152.42,141. 81,141.77,141.20,140.11,139.09,131.73,129.77,129.00,128.46,128.39, 127.46,126.81,126.31,122.87,119.51,118.91,114.83,113.00,109.80,106 .24,50.77,48.37,35.07,30.11,28.59,27.53,23.70,19.71.HR-MS(ESI):m / z calcd for C 42 H 40 N3O6S + :714.2632,found:714.2643.

[0103] The application test of the fluorescent probe prepared in this example is as follows:

[0104] 1) Preparation of stock solution for detection

[0105] a. Fluorescent probe sample solution (1.00×10 -3 mol / L) preparation: Accurately weigh 0.0073 g (M = 730) of fluorescent probe CYB-C1 and 0.0071 g (M = 714) of fluorescent probe CYB-C2, respectively, and dissolve them in 10 mL of dimethyl sulfoxide to a concentration of 1.00 × 10 -3 mol / L solution.

[0106] b. Chymotrypsin (Chy), trypsin, lysozyme, bovine serum albumin, lipase, pepsin, and cellulase were prepared in deionized water to a concentration of 1 mg / mL. AChE and BChE were prepared in deionized water to a concentration of 5 U / mL. Various amino acids and ions were prepared in deionized water to a concentration of 1.0 × 10 -2 mol / L solution.

[0107] The buffer solutions used in the following tests were all mixtures of PBS (10 mM, pH = 7.4) and DMSO in a volume ratio of 9:1, and the experimental water was all deionized water.

[0108] 2) Detection and analysis

[0109] 30 μL (1.00×10 -3 mol / L) probe stock solution was added to 3 mL of buffer and mixed evenly. The fluorescence excitation wavelength was 640 nm, the excitation slit width was 5.0 nm, the emission slit width was 5.0 nm, and the scanning range was set to 650-800 nm to obtain the fluorescence spectrum of the fluorescent probe. The fluorescence intensity of the system gradually increased with the increase of chymotrypsin concentration. The results are shown in FIG. Figure 8 (a) in Figure 8 and (c) in Figure 8. The fluorescence intensity values ​​(710 nm) detected at different concentrations were used to make the Figure 8 (b) and Figure 8 (d) shows a relationship between the two probes. Both probes can be fitted linearly at chymotrypsin concentrations of 2-5 μg / mL. The linear correlations obtained are 0.988 for CYB-C1 (with the fitting equation y = 129.7x + 598.59) and 0.991 for CYB-C2 (with the fitting equation y = 106.8x + 492.78). This indicates that within a certain range of chymotrypsin concentrations, the fluorescence intensity at 710.0 nm is positively correlated. The limit of detection (LOD) for chymotrypsin for both probes was calculated using the formula: LOD = 3σ / k, where σ is the standard deviation of five replicate blank measurements and k is the slope of the linear relationship between different cysteine ​​concentrations and the corresponding fluorescence intensity at 710 nm. The calculated limit of detection for chymotrypsin is 9.27 ng / mL for probe CYB-C1 and 13.46 ng / mL for probe CYB-C2.

[0110] 30 μL (1.00×10 -3 mol / L) probe stock solution was added to 3 mL of buffer and mixed evenly. The fluorescence excitation wavelength was 640 nm, the excitation slit width was 5.0 nm, the emission slit width was 5.0 nm, and the scanning range was set to 650-800 nm. The probe was first scanned alone to detect the fluorescence intensity at the maximum emission wavelength of 710 nm. Figure 9 (a) and Figure 9 As shown in (b), the probe remains stable in the buffer system. After the addition of chymotrypsin, the fluorescence emission at 710 nm of the probe solution is significantly enhanced. CYB-C1 can fully respond within 24 minutes, while CYB-C2 requires 42 minutes to fully respond.

[0111] 30 μL (1.00×10 -3 mol / L) probe stock solution was added to 3 mL of buffer and mixed evenly. The fluorescence excitation wavelength was 640 nm, the excitation slit width was 5.0 nm, the emission slit width was 5.0 nm, and the scanning range was set to 650-800 nm. The probe was scanned alone first to detect the fluorescence intensity at the maximum emission wavelength of 710 nm. Subsequently, the analytes (30 μL) (Chy, AChE, BChE, trypsin, lysozyme, bovine serum albumin, lipase, pepsin, cellulase, Ser, Glu, Met, Phe, Cys, GSH, Hys, Tyr, Lys, Leu, Mg) were added in sequence. 2+ 、Cu 2+ 、Zn 2+ 、Fe 2+ 、Co 2+ 、Gd 2+ 、F - 、NO2 - 、ClO4 - 、SCN - , detect the fluorescence intensity at the maximum emission wavelength of 710 nm. Figure 10 (a) is a comparison chart of the fluorescence intensity of the probe CYB-C1 and chymotrypsin at the maximum emission wavelength of 710 nm compared with other enzymes, amino acids, or ions. The fluorescence signal of CYB-C1 added to chymotrypsin is enhanced by approximately 6.5 times, the fluorescence signal of acetylcholinesterase (AChE) or butyrylcholinesterase (BChE) is enhanced by approximately 1.5 times, and the fluorescence signal of cysteine, homocysteine, or glutathione is enhanced by nearly 1 times. The fluorescence signal enhancement of other enzymes, amino acids, anions, and cations is less than 1 times. Figure 10 (b) is a comparison bar graph of the fluorescence intensity of the probe CYB-C2 and chymotrypsin at the maximum emission wavelength of 710 nm with other different enzymes, amino acids or ions. The fluorescence signal of CYB-C2 added to chymotrypsin is enhanced by about 6.5 times, while the fluorescence signal enhancement of other enzymes, amino acids, anions and cations is less than 1 times. In summary, CYB-C1 can respond to various active small molecules except chymotrypsin to varying degrees. In addition to responding to chymotrypsin, CYB-C2 has no obvious response to other active molecules, indicating that CYB-C2 has better selectivity than CYB-C1. Therefore, CYB-C2 was selected for subsequent biological activity verification.

[0112] MCF-7 cells were cultured in DMEM supplemented with 10 μg / mL Insulin and 10% FBS at 37°C and 5% CO2. The cultured MCF-7 cells were inoculated into confocal culture dishes, and confocal imaging experiments were performed after the cells adhered. The confocal culture dishes were numbered and grouped, and four groups of cells were treated and imaged differently. The first group of cells served as the blank group. The second group of cells were incubated with 5.0 μM CYB-C2 at 37°C for 45 minutes, washed three times with HBSS buffer solution, and then imaged. The third group of cells were pretreated with 10 μM phenylmethylsulfonyl fluoride (PMSF) at 37°C for 30 minutes, then incubated with 5 μM CYB-C2 for 45 minutes, and washed three times with HBSS buffer solution before imaging. The fourth group of cells was incubated with 30 μM phenylmethylsulfonyl fluoride (PMSF) at 37°C for 30 minutes, then incubated with 5 μM CYB-C2 probe for 45 minutes. They were washed three times with HBSS buffer solution before imaging. When fluorescence imaging was performed using a laser confocal microscope, the excitation wavelength was 638 nm and the collection range was 690-730 nm. The biological imaging image of the near-infrared fluorescent probe CYB-C2 detecting chymotrypsin in living cells is shown in Figure 2. Figure 11 As shown. Figure 11 As can be seen in group a, cells that were not incubated with the probe did not emit fluorescence. When MCF-7 cells were incubated with 5 μM probe at 37°C for 45 minutes, red fluorescence was observed. This fluorescence change indicates that the probe is sensitive to the natural level of chymotrypsin in living cells and can be used to monitor endogenous chymotrypsin ( Figure 11 To further confirm that the probe can specifically recognize chymotrypsin in living cells, group c was pretreated with PMSF (chymotrypsin inhibitor, 10 μM) for 30 minutes, and then incubated with 10 μM probe for 45 minutes before confocal imaging. Compared with group b treated with the probe alone, the fluorescence was weakened ( Figure 11 Group c). Group d was pretreated with PMSF (chymotrypsin inhibitor, 30 μM) for 30 min, and then incubated with 5 μM probe for 45 min before confocal imaging. As the inhibitor concentration increased, the fluorescence further weakened ( Figure 11 The above experimental results show that the near-infrared fluorescent probe CYB-C2 has good responsiveness and selectivity to chymotrypsin in cells and has good application prospects in biological testing.

[0113] To investigate the probe's ability to localize the Golgi apparatus, cultured MCF-7 cells were seeded onto confocal microplates and allowed to adhere before confocal imaging. MCF-7 cells were cultured in DMEM supplemented with 10 μg / mL insulin and 10% FBS at 37°C and 5% CO2. MCF-7 cells were treated with 5 μM probe and a commercial green Golgi-targeting dye (BODIPYTR Ceramide, 1 μg / mL) for 45 minutes, followed by imaging and localization analysis. Figure 12 As shown, the probe has strong tissue penetration and is capable of detecting chymotrypsin in cells. Furthermore, the red channel treated with the probe and the green channel treated with the Golgi-targeting dye overlap well, demonstrating good co-labeling performance with the Golgi-targeting dye. The Pearson correlation coefficient is 0.80, indicating that the probe can target the Golgi apparatus.

[0114] In order to further study the application of the probe, this application conducted an in vivo chymotrypsin imaging experiment on zebrafish. Zebrafish larvae will be cultured in E3 embryo culture medium (containing 0.15mM KCl, 0.33mM MgSO4, 0.33mM CaCl2 and 5mM NaCl, pH 7.0±1.0). Three-day-old zebrafish are divided into four groups and placed in glass dishes for experiments. The first group is a blank group, the second group is cultured with only 5mM CYB-C2 for 35 minutes; the third group is cultured with 10μM phenylmethylsulfonyl fluoride (PMSF) for 30 minutes, and then cultured with 5mM CYB-C2 for 35 minutes; the fourth group is cultured with 30μM PMSF for 30 minutes, and then cultured with 5mM CYB-C2 for 35 minutes. The excitation wavelength is 638nm, and the collection range is 690-730nm. As Figure 13 As shown, the blank group ( Figure 13 No red fluorescence was observed in group a), and the second group treated with CYB-C2 ( Figure 13 The zebrafish in group b) showed obvious red fluorescence, while the zebrafish in group 3 ( Figure 13 Group c) showed weak fluorescence. Group 4 ( Figure 13 In group d), the PMSF concentration increased, and the fluorescence produced was significantly weaker than that in group 3. The results showed that the probe can be used to detect chymotrypsin in vivo.

Claims

1. A Golgi apparatus-targeted near-infrared fluorescent probe for detecting chymotrypsin, wherein the structure of the fluorescent probe is as follows: Where R is selected from or , and R is When the compound is recorded as CYB-C1, R is When , the compound is recorded as CYB-C2.

2. The method for preparing the Golgi apparatus-targeted near-infrared fluorescent probe for detecting chymotrypsin according to claim 1, characterized in that: The following steps are involved: (1) Cyclohexanone, PBr3 and N,N-dimethylformamide react in dichloromethane to obtain compound I; (2) 2,4-dihydroxybenzaldehyde, 3,4-dihydro-2H-pyran and pyridinium p-toluenesulfonate react in dichloromethane to obtain compound II; (3) Compound I, compound II and potassium carbonate react in N,N-dimethylformamide to obtain compound III; (4) Compound III reacts with trifluoroacetic acid in dichloromethane with stirring to obtain compound IV; (5) p-Aminobenzenesulfonamide reacts with chloroacetyl chloride in acetone to obtain compound V; (6) Compound V reacts with 2,3,3-trimethyl-3H-indole in acetonitrile to obtain compound VI; (7) Compound IV, triethylamine and 4-bromobutyryl chloride react to obtain compound VII; (8) Compound IV, triethylamine and 3-phenylpropionyl chloride react to obtain compound VIII; (9) Compound VI reacts with compound VII in ethanol to obtain the fluorescent probe CYB-C1; (10) Compound VI reacts with compound VIII in ethanol to obtain the fluorescent probe CYB-C2; The structures of Compound I, Compound II, Compound III, Compound IV, Compound V, Compound VI, Compound VII and Compound VIII are as follows: 、 、 、 、 、 、 、 。 3. The method for preparing the Golgi apparatus-targeted near-infrared fluorescent probe for detecting chymotrypsin according to claim 2, characterized in that: Specifically include the following steps: (1) The preparation process of compound I is as follows: PBr3 is added dropwise to a mixed solution of N,N-dimethylformamide and dichloromethane under ice bath conditions, stirred for 0.5 h to 1 h, cyclohexanone is added to the mixed system, stirred for 24 h to 36 h, and after the reaction is complete, the mixture is neutralized to neutrality, the product is extracted with dichloromethane, and concentrated by rotary evaporation to obtain the target product; (2) The specific preparation process of compound II is as follows: 2,4-dihydroxybenzaldehyde, pyridinium p-toluenesulfonate, dichloromethane and 3,4-dihydro-2H-pyran are weighed and added to the reaction vessel in sequence, and after stirring and refluxing, the reaction is completed, followed by rotary evaporation and column chromatography to obtain compound II; (3) The preparation process of compound III is as follows: Compound I, compound II and potassium carbonate prepared above are added to DMF in sequence, stirred at 35-45°C for complete reaction, cooled to room temperature, filtered and concentrated to obtain a concentrated solution of compound III; (4) The preparation process of compound IV is as follows: the concentrated solution obtained in step (3) is dissolved in dichloromethane, trifluoroacetic acid is added to adjust the pH to 6-7, and the reaction is stirred at 35-45°C until the reaction is complete. The mixture is cooled to room temperature, filtered, washed with ethanol, and dried to obtain compound IV; (5) The preparation process of compound V is as follows: p-aminobenzenesulfonamide is weighed and added to acetone, chloroacetyl chloride is added dropwise, and the reaction is stirred at 70-80°C until complete, then cooled to room temperature, ice water is added, the resulting mixture is stirred, filtered, and the collected solid is washed with ice water, then recrystallized with ethanol, filtered, and the resulting solid is dried to obtain compound V; (6) The preparation process of compound VI is as follows: under nitrogen atmosphere, compound V obtained in step (5) is dissolved in acetonitrile, 2,3,3-trimethyl-3H-indole is added, and the reaction is refluxed until completion, the reaction solution is rotary evaporated, dichloromethane is added, and the reaction solution is filtered under reduced pressure to obtain a solid compound VI; (7) The preparation process of compound VII is as follows: under nitrogen atmosphere, compound IV and triethylamine are added to dichloromethane, 4-bromobutyryl chloride is added under ice bath condition, and after stirring, the reaction is completed, rotary evaporation and column chromatography are performed to obtain compound VII; (8) The preparation process of compound VII is as follows: under nitrogen atmosphere, compound IV and triethylamine are added to dichloromethane, 3-phenylpropionyl chloride is added under ice bath condition, and after stirring, the reaction is completed, rotary evaporation and column chromatography are performed to obtain compound VII; (9) The specific preparation process of the fluorescent probe CYB-C1 is as follows: under a nitrogen atmosphere, compound VII and compound VI are dissolved in ethanol, stirred and refluxed until a solid precipitate is formed, and then filtered. The obtained solid is washed with ethyl acetate and then subjected to column chromatography to obtain the probe CYB-C1; (10) The specific preparation process of the fluorescent probe CYB-C2 is as follows: under a nitrogen atmosphere, compound VIII and compound VI are dissolved in ethanol, stirred and refluxed until a solid precipitate is formed, and then filtered. The obtained solid is washed with ethyl acetate and then subjected to column chromatography to obtain the probe CYB-C2.

4. The method for preparing a Golgi apparatus-targeted near-infrared fluorescent probe for detecting chymotrypsin according to claim 2 or 3, characterized in that: In step (1), the molar ratio of PBr3, N,N-dimethylformamide and cyclohexanone is (2.5~3.0):(2.0~2.5):1; in step (2), the molar ratio of 2,4-dihydroxybenzaldehyde, 3,4-dihydro-2H-pyran and p-toluenesulfonic acid pyridinium salt is 1:(2.0~2.5):0.05; in step (3), the molar ratio of compound I, compound II and potassium carbonate is 1:1:3; in step (5), the molar ratio of p-aminobenzenesulfonamide and chloroacetyl chloride is 1:(1~1.5); in step (6), the molar ratio of compound V and 2,3,3-trimethyl-3H-indole is 1:(1~1.5); in step (7), the molar ratio of compound IV, triethylamine and 4-bromobutyryl chloride is 1:(1~2) :(1~2); in step (8), the molar ratio of compound IV, triethylamine and 3-phenylpropionyl chloride is 1:(1~2):(1~2); in step (9), the molar ratio of compound VI to compound VII is 1:(1~1.5); in step (10), the molar ratio of compound VIII to compound VI is 1:(1~1.5).

5. Use of the fluorescent probe according to claim 1 in the detection of chymotrypsin for non-disease diagnosis purposes.

6. Use of the fluorescent probe according to claim 1 in the preparation of a cell fluorescence imaging agent.

7. Use of the fluorescent probe according to claim 1 in preparing a fluorescent probe targeting the Golgi apparatus.

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