Chymotrypsin near-infrared fluorescent probe with large stokes shift and preparation method and application thereof

Abstract

The application discloses a chymotrypsin near-infrared fluorescent probe with a large stokes shift and a preparation method and application thereof. A fluorescent probe molecule TCF-CHT has a structural formula as shown in formula I. When a 530nm excitation wavelength is used for excitation, the TCF-CHT itself has weak fluorescence at 670nm, but it can selectively have a fluorescence opening reaction with the CHT. With the increase of the CHT concentration, the 670nm fluorescence intensity is gradually enhanced, and other common interfering ions and species cannot obviously interfere with and influence the TCF-CHT as an analysis reagent for detecting the CHT. Since the emission wavelength is in the near-infrared region and a large stokes shift (140nm) is generated, the interference of the system background fluorescence is greatly reduced, and the sensitivity of the CHT activity detection is improved. Therefore, the TCF-CHT is suitable for the fluorescence detection of the CHT with high selectivity and high sensitivity.

Description

Technical Field

[0001] This invention belongs to the field of analytical detection technology, specifically relating to a near-infrared fluorescent probe TCF-CHT for chymotrypsin with a large Stokes shift, its preparation method, and its application. Background Technology

[0002] Chymotrypsin (CHT) is a serine protease present in the human digestive system, playing a crucial role in maintaining physiological functions such as protein digestion, immune responses, and tissue repair. Studies have shown that abnormal CHT expression is closely linked to the pathogenesis of diabetes, hypertension, and various types of inflammation, particularly pancreatic cancer. Furthermore, CHT itself can be used as a drug to effectively prevent redness or damage caused by infection and surgery. Therefore, developing a selective, sensitive, and convenient real-time CHT detection technology is of great significance for CHT's application in drug development and clinical diagnosis.

[0003] Traditional methods for detecting CHT include enzyme-linked immunosorbent assay (ELISA), Western blotting, high-performance liquid chromatography (HLPC), and inductively coupled plasma mass spectrometry (ICP-MS). However, these methods suffer from drawbacks such as complex sample pretreatment, high cost, low spatiotemporal resolution, requirement for specialized personnel, and poor resistance to interference. Furthermore, peptide-based fluorescent substrates have been developed and used to measure CHT activity in vivo, but these substrates are susceptible to degradation and are unsuitable for long-term imaging. Compared to the above methods, molecular fluorescent probe technology offers unique advantages such as short response time, high sensitivity, convenience, ease of operation, non-invasiveness, and good biocompatibility, leading to its widespread application in biological and analytical chemistry fields. However, existing fluorescent probes for CHT detection suffer from drawbacks such as short emission wavelengths, susceptibility to interference from biological background fluorescence, long response times, and small Stokes shifts. Near-infrared fluorescent probes, with their strong tissue penetration and ability to effectively avoid biological background fluorescence interference, are more suitable for detecting CHT in vivo.

[0004] Therefore, it is particularly important to develop a near-infrared fluorescent probe with large Stokes shift characteristics that can be used to detect CHT activity. Summary of the Invention

[0005] One object of the present invention is to provide a near-infrared fluorescent probe molecule—TCF-CHT—that has large Stokes shift characteristics, high sensitivity and high selectivity for detecting CHT.

[0006] The TCF-CHT provided by this invention has the structural formula shown in Formula I:

[0007]

[0008] Another object of the present invention is to provide a method for preparing TCF-CHT as shown in Formula I.

[0009] The preparation method of TCF-CHT provided by this invention includes the following steps (see the preparation flowchart). Figure 1 ):

[0010] 1) React 5-bromothiophene-2-carboxaldehyde with p-hydroxyphenylboronic acid to generate compound 1;

[0011] 2) Reaction of malononitrile and 3-hydroxy-3-methyl-2-butanone produces compound 2;

[0012] 3) React compound 1 with compound 2 to generate TCF-OH;

[0013] 4) React compound TCF-OH with 4-bromobutyryl chloride to obtain TCF-CHT as shown in Formula I.

[0014]

[0015] In step 1) of the above method, the specific method for the reaction of 5-bromothiophene-2-carboxaldehyde with p-hydroxyphenylboronic acid is as follows:

[0016] 5-Bromothiophene-2-carboxaldehyde and p-hydroxyphenylboronic acid were dissolved in tetrahydrofuran, and then tetra(triphenylphosphine)palladium and a 22% (w / w) aqueous solution of potassium carbonate were added. The reaction was carried out under nitrogen protection and refluxed. The molar ratio of 5-bromothiophene-2-carboxaldehyde, p-hydroxyphenylboronic acid, and tetra(triphenylphosphine)palladium was 1:1:0.01. The reaction was carried out at 75°C for 3 hours.

[0017] In step 2) of the above method, the specific method for the reaction of malononitrile and 3-hydroxy-3-methyl-2-butanone is as follows: malononitrile, 3-hydroxy-3-methyl-2-butanone, and sodium ethoxide are dissolved in ethanol and heated under reflux. In the reaction, the molar ratio of 3-hydroxy-3-methyl-2-butanone, malononitrile, and sodium ethoxide is 1:3:0.15. The reaction temperature is 80℃, and the reaction time is 4 hours.

[0018] In step 3) of the above method, the specific method for the reaction of compound 1 and compound 2 is as follows: compound 1 and compound 2 are dissolved in anhydrous ethanol, piperidine is added, and the reaction is carried out under nitrogen protection by reflux. The molar ratio of compound 1, compound 2 and piperidine is 1:1:1.25. The reaction temperature is 80℃, and the reaction time is 3 hours.

[0019] In step 4) of the above method, the specific method for reacting the compound TCF-OH with 4-bromobutyryl chloride is as follows: TCF-OH and triethylamine are dissolved in dichloromethane, and 4-bromobutyryl chloride is added dropwise at 0°C. The mixture is then brought back to room temperature (25°C) and stirred to allow the reaction to proceed. In this reaction, the molar ratio of TCF-OH, 4-bromobutyryl chloride, and triethylamine is 1:1.2:1.5, and the reaction time is 3 hours.

[0020] Another object of the present invention is to provide the use of TCF-CHT.

[0021] The TCF-CHT application provided by this invention is selected from at least one of the following 1)-7):

[0022] 1) A fluorescent probe made of TCF-CHT;

[0023] 2) Application of TCF-CHT as a fluorescent probe or as a fluorescent probe for detecting CHT;

[0024] 3) Chemical sensors containing TCF-CHT;

[0025] 4) Application of TCF-CHT in the preparation of chemical sensors or chemical sensors for detecting CHT;

[0026] 5) Application of TCF-CHT in CHT detection;

[0027] 6) Application of the fluorescent probe in 1) above in the detection of CHT;

[0028] 7) Application of the chemical sensor described in 3) above in the detection of CHT;

[0029] 8) Application of TCF-CHT in the screening and / or evaluation of the inhibitory capacity of CHT inhibitors.

[0030] Both the fluorescent probe and the chemical sensor can be used for CHT detection and fluorescence imaging.

[0031] The fluorescent probe or chemical sensor can be applied to cells.

[0032] Specifically, the fluorescent probe or chemical sensor can be used for fluorescence imaging of endogenous CHT in mouse mast cell tumor cells (P815 cells) and human liver cancer cells (HepG2 cells).

[0033] The inventors of this invention have experimentally demonstrated that TCF-CHT can selectively react with CHT. Using 530 nm as the excitation wavelength, the fluorescence intensity at 670 nm gradually increases with increasing CHT concentration (0–100 μg / mL). The fluorescence emission intensity at 670 nm exhibits a good linear relationship with the CHT concentration, forming an excellent optical system with a large Stokes shift characteristic (140 nm) and good detection sensitivity and selectivity for CHT. Therefore, TCF-CHT is suitable for highly selective and sensitive detection of CHT, which can be performed using fluorescence spectroscopy.

[0034] When using fluorescence spectroscopy with TCF-CHT as the detection reagent to detect CHT, the method detection limit was 8.91 ng / mL, indicating that TCF-CHT has excellent sensitivity to CHT. Simultaneously, TCF-CHT exhibits excellent selectivity in its fluorescence response to CHT, effectively detecting common ions and interfering substances (such as Na+). + K + Mg 2+ Ca 2+ Fe 3+ S 2- NO2 - CO3 2- Cl - ,Br - I - The presence of ions (Cys, Gly, Arg, Ala, Lys, GSH, Phe, Met, His, Glu, H2O2, Pepsase, Tyrosinase, Lipase, Trypsin, Lysozyme, and Elastase) provides minimal interference to CHT determination, thus eliminating the influence of numerous interfering ions and species on the detection results, resulting in high detection specificity. Furthermore, when using TCF-CHT for CHT detection, the high sensitivity allows for the completion of the procedure with only a small sample volume, broadening the application range of this method. Attached Figure Description

[0035] Figure 1 This is a flowchart of the preparation process of TCF-CHT.

[0036] Figure 2 The 1H NMR spectrum of compound 1.

[0037] Figure 3 This is the carbon NMR spectrum of compound 1.

[0038] Figure 4 This is the high-resolution mass spectrum of compound 1.

[0039] Figure 5The 1H NMR spectrum of compound 2.

[0040] Figure 6 This is the carbon NMR spectrum of compound 2.

[0041] Figure 7 This is the high-resolution mass spectrum of compound 2.

[0042] Figure 8 The 1H NMR spectrum of TCF-OH is shown.

[0043] Figure 9 The image shows the carbon NMR spectrum of TCF-OH.

[0044] Figure 10 This is a high-resolution mass spectrometry of TCF-OH.

[0045] Figure 11 The TCF-CHT is a hydrogen nuclear magnetic resonance spectrum.

[0046] Figure 12 The image shows the carbon NMR spectrum of TCF-CHT.

[0047] Figure 13 This is a high-resolution mass spectrometry of TCF-CHT.

[0048] Figure 14 (a) and (b) are the UV absorption and fluorescence spectra of the TCF-CHT coexisting system with CHT (66.7 μg / mL) and the TCF-CHT and TCF-OH systems existing alone; (c) is the fluorescence spectrum of the TCF-CHT coexisting system with different concentrations of CHT; (d) is the linear relationship between the fluorescence intensity at 670 nm and the CHT concentration of the TCF-CHT coexisting system with different concentrations of CHT; (e) is the fluorescence intensity of the TCF-CHT coexisting system with different concentrations of CHT changing over time; (f) is the Michaelis-Menten equation linear relationship of the TCF-CHT coexisting system with different concentrations of CHT (66.7 μg / mL).

[0049] Figure 15 (a) Fluorescence intensity changes over time in the TCF-CHT and CHT (66.7 μg / mL) coexisting system at different temperatures; (b) Fluorescence intensity changes at different pH values ​​in the TCF-CHT and CHT (66.7 μg / mL) coexisting system and the TCF-CHT system alone.

[0050] Figure 16 (a) The fluorescence intensity changes in the coexistence systems of TCF-CHT, CHT, and different concentrations of benzyl sulfonyl fluoride (PMSF); (b) The inhibition rate of CHT by different concentrations of PMSF.

[0051] Figure 17This is a selectivity diagram of TCF-CHT for CHT detection.

[0052] Figure 18 The cell survival rate of P815 cells and HepG2 cells after incubation with different TCF-CHT concentrations is shown in the figure.

[0053] Figure 19 Laser confocal imaging of P815 cells for TCF-CHT detection of endogenous chymotrypsin.

[0054] Figure 20 Laser confocal imaging of HepG2 cells for TCF-CHT detection of endogenous chymotrypsin. Detailed Implementation

[0055] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0056] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0057] Example 1: Preparation of the chemical sensor molecule TCF-CHT

[0058] The reaction process is as follows Figure 1 As shown, the specific method is as follows:

[0059] 5-Bromothiophene-2-carboxaldehyde (1.188 mL, 10 mmol) and p-hydroxyphenylboronic acid (1.3793 g, 10 mmol) were added to 75 mL of tetrahydrofuran, followed by tetra(triphenylphosphine)palladium (100.0 mg, 0.1 mmol). Then, 20 mL of a 22% (w / w) potassium carbonate aqueous solution was rapidly added. The mixture was refluxed at 75 °C with stirring for 3 hours under nitrogen protection. After the reaction was complete, the mixture was filtered, and the filtrate was extracted with ethyl acetate and a saturated sodium chloride aqueous solution. The organic phase was dried over anhydrous sodium sulfate, and the organic solvent was removed by rotary evaporation to obtain a solid. The solid was purified by column chromatography using petroleum ether (boiling range 60–90 °C) / ethyl acetate (2:1, v / v) as the eluent to give a yellow solid compound 1 in 83% yield.

[0060] Malononitrile (3.96 g, 30 mmol), 3-hydroxy-3-methyl-2-butanone (1.02 g, 10 mmol), and sodium ethoxide (0.102 g, 1.5 mmol) were dissolved in 10 mL of ethanol and refluxed at 80 °C for 4 hours. After the reaction was complete, the mixture was cooled to room temperature (25 °C), and a white solid precipitated. The solid was filtered and the filter cake was washed with ice-cold ethanol to give compound 2 in 40% yield.

[0061] Compound 1 (94 mg, 0.4 mmol) and compound 2 (80 mg, 0.4 mmol) were dissolved in 6 mL of anhydrous ethanol, and then piperidine (50 μL, 0.5 mmol) was added. The mixture was refluxed and stirred at 80 °C for 3 hours under nitrogen protection. After the reaction was complete, the mixture was filtered to give a purple solid compound TCF-OH in 60% yield.

[0062] Compound TCF-OH (192.5 mg, 0.5 mmol) and triethylamine (110 mg, 0.75 mmol) were dissolved in 10 mL of dichloromethane. 4-Bromobutyryl chloride (77 mg, 0.6 mmol) was added dropwise at 0 °C, and the mixture was stirred for 10 min. After returning to room temperature, the reaction was allowed to proceed for 3 h. The reaction was quenched with 20 mL of distilled water, extracted with dichloromethane, dried over anhydrous Na₂SO₄, and the solvent was removed by rotary evaporation. The mixture was purified by column chromatography, with dichloromethane as the eluent, to give compound TCF-CHT in 30% yield.

[0063] NMR and high-resolution mass spectrometry identification results of compound 1: 1 H NMR (DMSO-d6, 500MHz) δ = 9.95 (s, 1H), 9.80 (s, 1H), 7.92 (d, J = 4.0Hz, 1H), 7.58 (d, J = 7.7Hz, 2H), 7.49 (d, J = 4.0Hz, 1H), 6.81 (d, J = 8.7Hz, 2H). 13 C10 NMR (DMSO-d6, 125MHz) δ=183.6,159.0,153.8,140.5,139.5,127.9(2C),123.5,123.4,116.1. The 1H and 1C NMR spectra are shown below. Figure 2 and Figure 3 Instrument model: Bruker Avance. HR-MS (ESI, m / z) cacld for C 11 H9O2S + [M+H] + :205.0323, found:205.0324. See results. Figure 4Instrument model: UPLC-Q / TOF Xevo G2-XS. The above results confirm that the obtained compound is indeed the target compound 1.

[0064] NMR and high-resolution mass spectrometry identification results of compound 2: 1 H NMR (CDCl3, 500MHz) δ = 2.36 (s, 3H); 1.62 (s, 6H). 13 C10 NMR (CDCl3, 125MHz) δ=182.8,175.3,111.1,110.5,109.1,104.9,99.9,58.5,24.4(2C),14.3. The 1H and 1C NMR spectra are shown below. Figure 5 and Figure 6 Instrument model: Bruker Avance. HR-MS (ESI, m / z) calibrated for C 11 H9N3O + [M+Na] + :222.0643, found:222.0644. See results. Figure 7 Instrument model: UPLC-Q / TOF Xevo G2-XS. The above results confirm that the obtained compound is indeed the target compound 2.

[0065] NMR identification results of TCF-OH: 1 H NMR (500MHz, DMSO-d6) δ = 10.05 (s, 1H), 8.11 (d, J = 15.9Hz, 1H); 7.79 (d, J = 4.0Hz, 1H); 7.64 (d, J =8.6Hz, 2H); 7.55 (d, J = 4.0Hz, 1H); 6.85 (d, J = 8.6Hz, 2H); 6.72 (d, J = 15.9Hz, 1H); 1.78 (s, 6H). 13 C10 NMR (DMSO-d6, 125MHz) δ=176.8,174.7,159.2,155.8,140.6,138.5,137.8,127.8(2C),124.4,123.6,116.2(2C),112.9,112.2,112.1,111.0,98.7,97.1,53.2,25.4(2C). The 1H and 1C NMR spectra are shown below. Figure 8 and Figure 9 Instrument model: Bruker Avance. HR-MS (ESI, m / z) calibrated for C 22 H 15 N3O2S + [M+Na] +:408.0783, found:408.0780. See results. Figure 10 Instrument model: UPLC-Q / TOF Xevo G2-XS. The above results confirm that the obtained compound is indeed the target compound TCF-OH.

[0066] NMR identification results of TCF-OH: 1 H NMR(DMSO-d6,500MHz)δ=8.12(d,J=16.1Hz,1H),7.86-7.83(m,3H),7.72(d,J=3.6Hz,1H),7.26(d,J=8.5H z,2H),6.82(d,J=8.7Hz,1H),3.63(t,J=6.6Hz,2H),2.76(t,J=7.2Hz,2H),2.21-2.16(m,2H),1.80(s,6H). 13 C10 NMR (DMSO-d6, 125MHz) δ=170.8,151.1,150.0,140.1,139.4,137.6,130.1,127.2(2C),126.3,122.8(2C),113.3(2C),112.7,111.9,110.8,98.9,98.3,54.8,53.8,33.8,32.1,27.5,25.3(2C). The 1H and 1C NMR spectra are shown below. Figure 11 and Figure 12 Instrument model: Bruker Avance. HR-MS (ESI, m / z) calibrated for C 26 H 20 BrN3O3S + [M+Na] + :556.0306, found:556.0303. See results. Figure 13 Instrument model: UPLC-Q / TOF Xevo G2-XS. The above results confirm that the obtained compound is indeed the target compound TCF-CHT.

[0067] Example 2: Fluorescence detection of CHT using TCF-CHT as an analytical reagent.

[0068] 1. Sensitivity of TCF-CHT for fluorescence detection of CHT

[0069] Weigh an appropriate amount of TCF-CHT and dissolve it in dimethyl sulfoxide (DMSO) to prepare a 1.0 mM stock solution, labeled as TCF-CHT stock solution. Dissolve CHT in a certain amount of phosphate buffer solution (PBS, 10 mM, pH = 7.4) to prepare a 3 mg / mL stock solution, labeled as CHT stock solution.

[0070] Add 8 μL of TCF-CHT stock solution and an appropriate volume of CHT stock solution to a 5 mL plastic EP tube. Then add an appropriate amount of phosphate buffered saline (PBS) / dimethyl sulfoxide (DMSO) mixture (6:4, v / v, PBS 10.0 mM, pH = 7.4) to achieve CHT concentrations of 0, 3.3, 6.7, 10, 13.3, 16.6, 20, 26.7, 33.3, 40, 46.7, 53.3, 60, 66.7, 73.3, 80, 86.7, 93.3, and 100 μg / mL in each test system, with a TCF-CHT concentration of 2 μM. After incubation at 37 °C for 30 minutes, transfer the system to a 1 cm quartz cell and measure the UV absorption and fluorescence spectra of the reaction system.

[0071] Figure 14 Images (a) and (b) show the UV absorption and fluorescence spectra of TCF-CHT and CHT (66.7 μg / mL) and TCF-OH and TCF-CHT alone. The excitation wavelength was 530 nm and the emission wavelength was 670 nm. Figure 14 As shown in (a), the maximum absorption wavelength of TCF-CHT is at 500 nm. After the addition of CHT, the UV absorption wavelength of the system red-shifts to 530 nm, and basically coincides with the UV absorption spectrum of the fluorescent parent TCF-OH; from Figure 14 As can be seen in (b), TCF-CHT itself has no obvious fluorescence at 670 nm. When CHT is added to the system, the fluorescence intensity of the system at 670 nm increases significantly and basically coincides with the fluorescence emission spectrum of the fluorescent parent TCF-OH. Figure 14 Figures (c) and (d) show the fluorescence spectra of TCF-CHT coexisting with different concentrations of CHT (0-100 μg / mL) and the linear relationship between fluorescence intensity at 670 nm and CHT concentration. Figure 14 As shown in (c) and (d), the fluorescence intensity of the reaction system at 670 nm gradually increases with the increase of CHT concentration. In the range of 0-20 μg / mL, the CHT concentration and the fluorescence intensity of the system are linearly related, and the linear equation is Y = 6.533X + 68.411. The detection limit of the method is calculated by dividing the standard deviation of the blank signal by 3 times by the slope of the standard curve, which is 8.91 ng / mL. Figure 14 (e) in the figure shows the fluorescence intensity change over time in the TCF-CHT coexisting system with different concentrations of CHT. Figure 14As shown in (e), at different CHT concentrations (0 μg / mL, 16.7 μg / mL, 33.3 μg / mL, 50 μg / mL, and 66.7 μg / mL), the fluorescence intensity of the TCF-CHT coexisting system gradually increased with time. When the CHT concentration was 66.7 μg / mL, the fluorescence intensity of the system basically stabilized after 30 minutes. Figure 14 Figure (f) shows the linear relationship of the Michaelis-Menten equation for the coexistence systems of different concentrations of TCF-CHT and CHT (66.7 μg / mL). The maximum enzymatic reaction rate (VL) was obtained based on the Michaelis-Menten equation. max The flow rate is 0.75 μM / min, and the Michaelis constant (k) is... m The concentration was 10.45 μM, indicating that CHT has a strong binding and catalytic ability to TCF-CHT.

[0072] Figure 15 Figure (a) shows the fluorescence intensity of the TCF-CHT and CHT (66.7 μg / mL) coexisting system over time at different temperatures. Figure 15 As can be seen from (a) in the figure, at four different temperatures (25℃, 30℃, 37℃ and 45℃), the fluorescence intensity of the TCF-CHT and CHT (66.7μg / mL) coexisting system gradually increases with time. At the same time, the fluorescence intensity of the system at 37℃ is the highest, indicating that 37℃ is the optimal reaction temperature for TCF-CHT and CHT. Figure 15 Figure (b) shows the fluorescence intensity trends at different pH values ​​for the coexistence of TCF-CHT and CHT (66.7 μg / mL) and the system containing TCF-CHT alone. Figure 15 As shown in (b), when the pH is in the range of 4.0–10.0, TCF-CHT alone exhibits no obvious fluorescence at 670 nm, and its fluorescence intensity hardly changes with pH. When CHT is added to the system, the fluorescence intensity is highest at pH = 6.0, and there is still a relatively high fluorescence intensity in the system at pH = 7.4, indicating that TCF-CHT is suitable for the fluorescence recognition of CHT under physiological conditions.

[0073] Figure 16 Figures (a) and (b) show the fluorescence intensity changes and the inhibition effect of different concentrations of PMSF on CHT in the coexistence systems of TCF-CHT, CHT, and phenylmethylsulfonyl fluoride (PMSF). Figure 16As shown in (a) and (b), the fluorescence intensity of the reaction system at 670 nm gradually decreases with increasing PMSF concentration (0-1 mM), and the relative activity of CHT decreases with increasing PMSF concentration. The degree of inhibition was determined using (F0-F1) / (F0-F2), where F0 represents the fluorescence intensity in the absence of PMSF, F1 represents the fluorescence intensity in the presence of PMSF, and F2 represents the fluorescence intensity in the absence of both PMSF and CHT. Based on the correlation between inhibition efficiency and PMSF concentration, the IC50 was determined. 50 The value was 0.55 mM. Therefore, the probe TCF-CHT shows great promise in the evaluation and screening of CHT inhibitors.

[0074] The above results indicate that the analytical reagent—TCF-CHT—has excellent fluorescence properties and can achieve highly sensitive fluorescence detection of CHT.

[0075] 2. Specificity of TCF-CHT for fluorescence detection of CHT

[0076] Simultaneously, select several 5mL EP tubes and perform similar operations as described above, except that instead of adding CHT, add various common interfering ions or substances. Samples 1 to 29 correspond to: Na + K + Mg 2+ Ca 2+ Fe 3+ S 2- NO2 - CO3 2- Cl - ,Br - I - Cys, Gly, Arg, Ala, Lys, GSH, Phe, Met, His, Glu, H2O2 (interfering ion or substance concentration of 100 μM), Pepsase, Tyrosinase, Lipase, Trypsin, Lysozyme, Elastase (interfering enzyme concentration of 150 U / mL), and no interfering substance (blank). Test results are shown below. Figure 17 .from Figure 17As can be seen, before the addition of CHT, the fluorescence intensity of the system did not change significantly when only the probe TCF-CHT and different types of interfering ions or substances were present, indicating that the probe TCF-CHT did not have a significant fluorescence response to the aforementioned interfering ions or substances. Subsequently, when CHT (66.7 μg / mL) was added to the systems 1–29 above, the fluorescence intensity at 670 nm increased significantly, indicating that the probe TCF-CHT has a good response to CHT, and the presence of the aforementioned interfering ions and substances will not significantly interfere with or affect the detection of CHT by TCF-CHT as an analytical reagent. Therefore, as a detection reagent, TCF-CHT has high selectivity and anti-interference ability for the fluorescence detection of CHT.

[0077] 3. Performance comparison of TCF-CHT with other CHT fluorescent probes

[0078] The fluorescence detection performance of TCF-CHT for CHT was summarized and compared with that of other fluorescent probes for CHT detection in the literature. The results are shown in Table 1. Table 1 shows that when using TCF-CHT for CHT fluorescence detection, the emission wavelength is in the near-infrared region (670 nm) and has a large Stokes shift (140 nm), which effectively reduces background fluorescence interference and thus improves detection sensitivity. The overall performance of TCF-CHT for CHT fluorescence detection is superior to that of similar probes reported previously.

[0079] Table 1. Performance comparison of TCF-CHT and CHT fluorescent probes in the literature.

[0080]

[0081]

[0082] References

[0083] [1]Zi YQ,Xu DW,Li C.,Qu F.,Zhao

[0084] [2]Wu L.,Yang S.H.,Xiong H.,Yang J.Q.,Guo J.,Yang W.C.,YangG.F.Nonpeptide-BasedSmall-Molecule Probe for Fluorogenic and ChromogenicDetection of Chymotrypsin.Analytical Chemistry.2017,89,3687-3693.

[0085] [3]Okorochenkova Y.,Porubsky M.,Benicka S.Hlavac J.A novel three-fluorophore system as aratiometric sensor for multiple protease detection.!Chemical Communication.2018,54,7589-7592.

[0086] [4]Wang J.M.,Teng Z.D.,Cao T.,Qian J.,Lei Z.,Cao Y.P.,Qin W.W.,GuoH.C.Turn-onvisible and ratiometric near-infrared fluorescent probes fordistinction endogenous esterasesand chymotrypsins in live cells.Sensors andActuators B:Chemical.2020,306,127567.

[0087] [5] Liu SY, Zou XT, Gao X., Zhang YYDe Novo design of a highly selective nonpeptide fluorogenic probe for chymotrypsin activity sensing in aliving system. Analytical Chemistry. 2022, 94, 17922-17929. Example 3: Fluorescence imaging of CHT in cells using TCF-CHT as an analytical reagent. Mouse mast cell tumor cells (P815 cells) and human liver cancer cells (HepG2 cells) were used as research subjects to carry out the cytotoxicity CCK-8 experiment of TCF-CHT. Cells were treated with different concentrations of TCF-CHT (0-50 μM) and cultured for 24 hours before the CKK-8 experiment was performed. The results are shown in the figure. Figure 18 .from Figure 18 As shown in (a) and (b), the cell viability is greater than 80%, indicating that TCF-CHT has low cytotoxicity against mouse mast cell tumor cells (P815 cells) and human liver cancer cells (HepG2 cells), making it suitable for cell imaging studies. Fluorescence imaging of endogenous CHT in mouse mast cell tumor cells (P815 cells) and human liver cancer cells (HepG2 cells) was performed using the probe TCF-CHT. The culture and imaging procedures for P815 and HepG2 cells were as follows: P815 cells and HepG2 cells (2×10⁶ cells) were cultured and imaged at 37°C. 4 Cells (cells / mL) were placed in sterile culture dishes and cultured in DMEM medium containing 10% embryonic serum for 24 h, maintaining a 5% CO2 atmosphere. Cells were then transferred to 96-well plates and incubated overnight. After washing twice with PBS buffer (pH 7.4), images were taken as a blank control group. Figure 19 (a) and Figure 20 (a) Then, fresh, complete culture medium (phenol red-free) preheated to 37°C was added to the above system, followed by a certain volume of TCF-CHT solution to ensure a final TCF-CHT concentration of 10 μM. Laser confocal microscopy was immediately performed, with images taken and recorded every 30 minutes as a probe group. The results are shown in (a). Figure 19 (b) and Figure 20 (b) Under the same conditions, cells were incubated with a solution of the chymotrypsin inhibitor PMSF (final concentration 500 μM) for 0.5 h, followed by the addition of a certain volume of TCF-CHT solution to ensure a final TCF-CHT concentration of 10 μM. Laser confocal microscopy was then performed immediately, with images taken every 30 minutes as the inhibitor group. The results are shown in (b). Figure 19(c) and Figure 20 (c) in the example. Figure 19 (a) and Figure 20 As shown in (a): P815 cells and HepG2 cells did not exhibit fluorescence before the addition of TCF-CHT. Figure 19 (b) and Figure 20 (b) shows that after co-incubation with TCF-CHT (10 μM) in P815 and HepG2 cells, the fluorescence of the red channel gradually increased over time. Figure 19 (c) and Figure 20 The results in (c) indicate that P815 and HepG2 cells pretreated with PMSF (500 μM) showed no significant fluorescence after co-incubation with TCF-CHT (10 μM), suggesting that chymotrypsin activity was almost completely inhibited by PMSF, and the fluorescence signal was mediated by CHT. Furthermore, the fluorescence enhancement of TCF-CHT was more pronounced in HepG2 cells than in P815 cells, indicating that CHT activity was higher in HepG2 cells than in P815 cells. These results clearly demonstrate that the TCF-CHT probe can be used for fluorescence imaging of endogenous chymotrypsin.

Claims

1. A compound TCF-CHT represented by Formula I: ###0001### 2. A method for preparing the compound TCF-CHT represented by Formula I in claim 1, comprising the following steps: 1) reacting 5-bromothiophene-2-carboxaldehyde with p-hydroxybenzoic acid to form compound 1; 2) reacting malononitrile and 3-hydroxy-3-methyl-2-butanone to form compound 2; 3) reacting compound 1 with compound 2 to form TCF-OH; 4) reacting compound TCF-OH with 4-bromobutyryl chloride to obtain TCF-CHT represented by Formula I; wherein the step 1), the specific method for reacting 5-bromothiophene-2-carboxaldehyde with p-hydroxybenzoic acid is as follows: dissolving 5-bromothiophene-2-carboxaldehyde and p-hydroxybenzoic acid in tetrahydrofuran, then adding tetrakis(triphenylphosphine)palladium and 22% mass fraction potassium carbonate aqueous solution, and refluxing under nitrogen protection; in the reaction, the molar ratio of 5-bromothiophene-2-carboxaldehyde, p-hydroxybenzoic acid and tetrakis(triphenylphosphine)palladium is 1:1:0.01, the reaction temperature is 75℃, and the reaction time is 3 hours; or, in the step 2), the specific method for reacting malononitrile and 3-hydroxy-3-methyl-2-butanone is as follows: dissolving malononitrile, 3-hydroxy-3-methyl-2-butanone and sodium ethoxide in ethanol, and refluxing under heating; in the reaction, the molar ratio of 3-hydroxy-3-methyl-2-butanone, malononitrile and sodium ethoxide is 1:3:0.15, the reaction temperature is 80℃, and the reaction time is 4 hours; or, in the step 3), the specific method for reacting compound 1 with compound 2 is as follows: dissolving compound 1 and compound 2 in anhydrous ethanol, adding piperidine, and refluxing under nitrogen protection; in the reaction, the molar ratio of compound 1, compound 2 and piperidine is 1:1:1.25, the reaction temperature is 80℃, and the reaction time is 3 hours; or, in the step 4), the specific method for reacting compound TCF-OH with 4-bromobutyryl chloride is as follows: dissolving compound TCF-OH and triethylamine in dichloromethane, adding 4-bromobutyryl chloride dropwise at 0℃, and stirring under room temperature; in the reaction, the molar ratio of compound TCF-OH, 4-bromobutyryl chloride and triethylamine is 1:1.2:1.5, and the reaction time is 3 hours. The fluorescent probe is the compound TCF-CHT in claim 1. The chemical sensor contains the compound TCF-CHT in claim 1.

6. The compound TCF-CHT in claim 1 or the fluorescent probe in claim 4 or the chemical sensor in claim 5 is applied in detecting CHT or in CHT fluorescence imaging; the application is non-disease diagnosis or treatment purpose.

7. The compound TCF-CHT in claim 1 or the fluorescent probe in claim 4 or the chemical sensor in claim 5 is applied in screening and / or inhibitory capacity evaluation of CHT inhibitors; the application is non-disease diagnosis or treatment purpose.

3. The method of claim 2, wherein: ​ ​ ​ ​ 4. A fluorescent probe characterized by: ​ 5. A chemical sensor characterized by: ​ ​ ​ 8. Use of the compound TCF-CHT according to claim 1 in at least one of 1) - 2): 1) as a fluorescent probe or as a fluorescent probe for detecting CHT; 2) in the preparation of a chemical sensor or a chemical sensor for detecting CHT; said use being a use other than for diagnostic or therapeutic purposes. The object of detection by said fluorescent probe or chemical sensor is a cell. ​ ​ ​ 9. Use according to claim 8, characterized in that: ​

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