A ratio type near-infrared fluorescent probe based on ICT-FRET mechanism and a preparation method and application thereof

Abstract

The application discloses a ratio type near-infrared fluorescent probe based on an ICT-FRET mechanism and a preparation method and application thereof. The ratio type near-infrared fluorescent probe based on the ICT-FRET mechanism emits in a near-infrared region, has an ultra-large stokes shift, can effectively avoid excitation light interference and self-absorption, reduces biological background fluorescence, and is suitable for deep tissue imaging. The probe can realize double-emission ratio response (I 660 / I 540 ) based on the FRET mechanism, has a significant signal change, has inherent self-calibration capability, shows extremely high selectivity to Cys, has almost no response to Hcy and GSH similar in structure, is strong in anti-environmental interference, and is more accurate in quantification. The TPP targeting group of the probe enables the probe to be specifically positioned in mitochondria of living cells, realizes dynamic ratio imaging of endogenous and exogenous Cys in mitochondria, and provides a powerful tool for studying Cys metabolism at a subcellular level.

Description

Technical Field

[0001] This invention belongs to the fields of analytical chemistry, biosensing and food safety detection technology, specifically relating to a ratiometric near-infrared fluorescent probe based on the ICT-FRET mechanism, its preparation method and application. Background Technology

[0002] Cysteine ​​(Cys), an essential amino acid containing a thiol group, plays a crucial role in maintaining redox homeostasis, protein structure and function, and as a precursor to glutathione. Cys is also an important nutrient and common food additive (such as an antioxidant and dough improver). Therefore, developing rapid and accurate methods for detecting Cys is of great significance for life science research, disease diagnosis, and food safety monitoring.

[0003] Currently, conventional methods for detecting Cys include high-performance liquid chromatography (HPLC), electrochemical methods, and capillary electrophoresis. While these methods are accurate, they typically require large instruments, complex sample pretreatment, and specialized personnel, making real-time, on-site, and in-situ detection difficult. Fluorescent probe technology, with its advantages of high sensitivity, ease of operation, and real-time imaging, has become a research hotspot. Numerous fluorescent probes for Cys detection have been reported to date.

[0004] However, most existing Cys fluorescent probes still have the following technical limitations: 1. Single signal mode: Most probes are "turn-on / off" type, and their fluorescence intensity is easily affected by probe concentration, environmental factors, instrument efficiency, etc., which affects the accuracy of quantification.

[0005] 2. Poor optical performance: Most probes emit wavelengths in the visible light region (<650nm), resulting in shallow tissue penetration and strong background fluorescence. Furthermore, the small Stokes shift easily leads to excitation light scattering interference and self-absorption.

[0006] 3. Insufficient differentiation ability: The selectivity for distinguishing structurally similar biothiols, such as homocysteine ​​(Hcy) and glutathione (GSH), is not ideal.

[0007] 4. Limited functionality: Most probe designs focus only on solution detection or cell imaging, lacking an integrated, multi-modal application platform that combines biomedical diagnostics with rapid on-site food safety testing.

[0008] 5. Lack of targeting: Few probes can specifically target organelles (such as mitochondria) for subcellular localization imaging of Cys, which limits its application in the study of fine biological processes such as cell metabolism.

[0009] Therefore, there is an urgent need in this field to develop an innovative Cys fluorescent probe that combines ratiometric signal self-calibration, near-infrared emission and large Stokes shift, high selectivity (especially for Hcy / GSH), mitochondrial targeting capability, and compatibility with both bioimaging and rapid on-site detection requirements. Summary of the Invention

[0010] The purpose of this invention is to provide a ratiometric near-infrared fluorescent probe based on the ICT-FRET mechanism, its preparation method, and its applications. This probe, based on the ICT-FRET synergistic mechanism, achieves a highly selective and sensitive ratiometric fluorescence response to Cys and possesses mitochondrial targeting capability.

[0011] To achieve the above objectives, the present invention provides the following technical solution: One of the technical solutions of this invention is to provide a ratiometric near-infrared fluorescent probe based on the ICT-FRET mechanism, with the following structural formula: .

[0012] The design concept of the ratiometric near-infrared fluorescent probe based on the ICT-FRET mechanism in this invention is as follows: Construction of the FRET pair: A naphthalimide-piperazine unit (NP) was used as the energy donor, and a cyanoisophorone derivative (DCI) was used as the energy acceptor. The emission spectra of the donor and the absorption spectra of the acceptor effectively overlapped, laying the foundation for the FRET process.

[0013] Specific recognition group: An acrylate group is introduced onto the DCI acceptor unit as a specific reaction site for Cys. This group acts as a "fluorescence quenching switch" in the initial state, inhibiting the intramolecular charge transfer (ICT) process and its fluorescence in the DCI unit.

[0014] Mitochondrial targeting group: Introduction of triphenylphosphine cation (TPP) into the molecule + The negative potential of the mitochondrial membrane is used to achieve mitochondrial-specific enrichment of the probe.

[0015] Connection and Regulation: The donor and acceptor are covalently linked via a piperazine ring, forming a rigid FRET backbone. The introduction and removal of acrylate directly regulate the receptor's ICT effect and the donor-to-receptor FRET efficiency.

[0016] The reaction mechanism between the probe DCINP and Cys is as follows: the thiol group of Cys first undergoes Michael addition to the acrylate group, followed by an intramolecular amino group attacking the ester group to undergo a cyclization-elimination reaction, cleaving the acrylate bond and releasing a phenoxy anion (DCINP-O) with strong electron-donating ability. -This structural shift produces two key photophysical effects: ICT effect activation: Phenoxy anions greatly enhance the "push-pull" electronic structure of the DCI unit, activating its ICT process and causing a redshift in the absorption and emission spectra.

[0017] FRET channel restoration: DCI is activated as a highly fluorescent receptor, and at the same time, the efficient FRET channel from the donor NP to the receptor DCI is opened.

[0018] Ultimately, under single-wavelength excitation (approximately 409 nm), the probe's fluorescence emission shifted from predominantly at 540 nm (green, NP donor emission) to predominantly at 660 nm (near-infrared red, DCI acceptor emission), achieving a significant ratiometric signal change (IL). 660 / I 540 (A significant increase).

[0019] The second technical solution of the present invention provides a method for preparing the above-mentioned ratiometric near-infrared fluorescent probe based on the ICT-FRET mechanism, comprising the following steps: Starting with 4-bromo-1,8-naphthalenedicarboxylic anhydride and β-alanine, a carboxylic acid intermediate was generated through a condensation reaction. The carboxylic acid intermediate was then reacted with thionyl chloride to generate an acyl chloride intermediate. The acyl chloride intermediate was then reacted with a triphenylphosphine derivative via an amidation reaction to obtain a triphenylphosphine-modified naphthalenedicarboxylic anhydride intermediate. The triphenylphosphine-modified naphthalenedicarboxylic anhydride intermediate was then subjected to a nucleophilic substitution reaction with 1-Boc-piperazine, followed by removal of the Boc protecting group and introduction of a piperazine linker to obtain an amino intermediate. The amino intermediate and the cyanoisophorone derivative were covalently linked by a reductive amination reaction to obtain the precursor compound (DCINP-OH); the precursor compound was reacted with acryloyl chloride under alkaline conditions to obtain the ratiometric near-infrared fluorescent probe based on the ICT-FRET mechanism. The structural formula of the carboxylic acid intermediate is as follows: ; The structural formula of the triphenylphosphine derivative is: ; The structural formula of the triphenylphosphine-modified naphthalenedicarboximide intermediate is as follows: ; The structural formula of the amine intermediate is: ; The structural formula of the cyanoisophorone derivative is: ; The structural formula of the precursor compound is: .

[0020] The third technical solution of the present invention provides an application of the above-mentioned ratiometric near-infrared fluorescent probe based on the ICT-FRET mechanism in the preparation of reagents for fluorescent imaging of cysteine ​​in mitochondria of cells or live animals.

[0021] The fourth technical solution of the present invention provides an application of the above-mentioned ratiometric near-infrared fluorescent probe based on the ICT-FRET mechanism in the preparation of reagents or kits for the detection of cysteine ​​in food.

[0022] Fifth technical solution of the present invention: to provide an application of the above-mentioned ratiometric near-infrared fluorescent probe based on the ICT-FRET mechanism in the preparation of a cysteine ​​detection kit.

[0023] The sixth technical solution of this invention: provides a method for quantitative detection of cysteine ​​based on an RGB analysis system, comprising the following steps: The ratiometric near-infrared fluorescent probe based on the ICT-FRET mechanism was mixed with different concentrations of cysteine ​​and photographed under a UV lamp. The RGB values ​​of the photographs were extracted using an RGB analysis system. A standard curve was established using the red / green channel intensity ratio of the obtained RGB values ​​and the corresponding cysteine ​​concentration as parameters. The sample to be tested was mixed with the ratiometric near-infrared fluorescent probe based on the ICT-FRET mechanism and photographed under a UV lamp. The RGB values ​​of the photographs were extracted using an RGB analysis system. The cysteine ​​content of the sample to be tested was calculated based on the standard curve.

[0024] The beneficial technical effects of the present invention are as follows: Superior optical performance: The ratiometric near-infrared fluorescent probe provided by this invention emits in the near-infrared region (660 nm) and has an ultra-large Stokes shift (251 nm), which can effectively avoid excitation light interference and self-absorption, reduce biological background fluorescence, and is suitable for deep tissue imaging.

[0025] High-precision ratio detection: The ratiometric near-infrared fluorescent probe provided by this invention can achieve dual emission ratio response (I0) based on the FRET mechanism. 660 / I 540 The signal changes significantly (~60 times enhanced), it has inherent self-calibration capability, strong resistance to environmental interference, and more accurate quantification.

[0026] Excellent selectivity and sensitivity: The ratiometric near-infrared fluorescent probe provided by this invention exhibits extremely high selectivity for Cys and almost no response to structurally similar Hcy and GSH. The kinetic constants show that its reaction rate with Cys is 13.6 times and 25.2 times that of Hcy and GSH, respectively, and the detection limit is as low as 37 nM.

[0027] Successful mitochondrial-targeted imaging: The TPP targeting group of the ratiometric near-infrared fluorescent probe provided by this invention enables the probe to be specifically located in the mitochondria of living cells, realizing dynamic ratio imaging of endogenous and exogenous Cys in mitochondria, and providing a powerful tool for studying Cys metabolism at the subcellular level.

[0028] In vivo imaging capability: The ratiometric near-infrared fluorescent probe provided by this invention has been successfully applied to ratiometric fluorescence imaging of Cys in mice, verifying its application potential in complex biological systems.

[0029] An innovative dual-mode application platform: The ratiometric near-infrared fluorescent probe provided by this invention can not only be used for precision laboratory instrument detection, but also successfully constructs a portable, visualized on-site detection platform based on smartphone RGB analysis. This platform can rapidly and accurately quantify the Cys content in various foods (such as garlic, chili peppers, and tomatoes), with results highly consistent with standard HPLC methods, achieving a seamless connection from basic research to rapid on-site detection. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 The diagram shows the design principle and reaction mechanism of the probe DCINP (a), the donor / acceptor spectral overlap diagram (b), and the fluorescence spectrum comparison diagram before and after the reaction (c).

[0032] Figure 2 For the various potential interfering substances in Example 2 and Cys' I 660 / I 540 ratio.

[0033] Figure 3 The fluorescence intensity curves are those obtained by adding different concentrations of Cys to the DCINP solution in Example 2.

[0034] Figure 4 In Example 2, I was obtained by adding different concentrations of Cys to the DCINP solution. 660 / I 540ratio.

[0035] Figure 5 The image shows the confocal imaging results of mitochondrial colocalization in Example 3; where a is the green channel fluorescence image of DCINP, b is the red channel fluorescence image of MitoTracker Red, c is the bright field image, d is the overlay of a, b, and c, e is the colocalization scatter plot of DCINP and MitoTracker Red fluorescence, and f is the fluorescence intensity distribution curve along the selected white arrow direction.

[0036] Figure 6 The results of ratio fluorescence imaging (a) and quantitative analysis (b) of probe DCINP in the mitochondria of live cells (HepG2) in Example 3 are shown.

[0037] Figure 7 The results of ratio fluorescence imaging of exogenous Cys in mice in vivo by probe DCINP in Example 4 are shown in (a) and (b) in red / green fluorescence intensity ratio.

[0038] Figure 8 The diagram shows the food sample pretreatment process in Example 5 (a), and the physical image of the portable detection platform based on a smartphone, the standard curve, and the sample detection results (b). Detailed Implementation

[0039] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0040] It should be noted that any aspects not described in detail in this invention are conventional practices in the field and are not the focus of this invention.

[0041] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0042] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this invention.

[0043] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0044] Unless otherwise specified, room temperature in this invention refers to a temperature of 20±10℃.

[0045] Example 1 Synthesis of the target probe DCINP: (1) Synthesis of intermediate 3: Following the methods described in known literature (Yang, Y.; Ma, M.; Shen, L.; An, J.; Kim, E.; Liu, H.; Jin, M.; Wang, S.; Zhang, J.; Kim, JS; Yin, C. A Fluorescent Probe for Investigating the Role of Biothiols in Signaling Pathways Associated with Cerebral Ischemia-Reperfusion Injury. Angew. Chem. Int. Ed. 2023, 62,e202310408. DOI: 10.1002 / anie.202310408.), intermediate 3 was prepared by Knoevenagel condensation reaction using isophorone and malononitrile as starting materials under the catalysis of an organic base piperidine, as detailed below: Isophorone (4.146 g, 30 mmol) and malononitrile (1.9820 g, 30 mmol) were dissolved in 100 mL of ethanol, and 0.5 mL of piperidine was added as a catalyst and the mixture was heated to reflux. The reaction progress was monitored by thin-layer chromatography. After the reaction was complete, the mixture was cooled and extracted with dichloromethane. The collected organic layers were combined, dried over anhydrous sodium sulfate, concentrated under vacuum, and finally purified by silica gel column chromatography to give colorless crystalline intermediate 1 (1.284 g, yield: 23%).

[0046] Equimolar amounts of 3-bromo-4-hydroxybenzaldehyde (1.45 g, 6.9 mmol) and intermediate 1 (1.28 g, 6.9 mmol) were dissolved in 20 mL of acetonitrile. Five drops of piperidine were added as a catalyst, and the mixture was heated to reflux. The reaction progress was monitored by thin-layer chromatography. After the reaction was complete, the mixture was cooled and extracted with dichloromethane. The collected organic layers were combined, dried over anhydrous sodium sulfate, concentrated under vacuum, and finally purified by silica gel column chromatography (eluent: dichloromethane) to give yellow intermediate 2 (0.74 g, yield: 29%).

[0047] Intermediate 2 (0.72 g, 2 mmol) and hexamethylenetetramine (HMTA, 0.56 g, 4 mmol) were dissolved in trifluoroacetic acid (10 mL) and refluxed at 73 °C with stirring for 3 hours. After cooling to room temperature, the reaction mixture was poured into ice water and extracted with dichloromethane. The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. After removing the solvent under reduced pressure, the crude product was purified by rapid column chromatography (eluent: dichloromethane) to give intermediate 3 (0.18 g, 45% yield), an orange-yellow solid.

[0048] The reaction process in step (1) is as follows: (2) Synthesis of intermediate 8: 1. Synthesis of intermediate 4: Intermediate 4 was synthesized according to the methods described in the literature (Kaur, A.; Brigden, KWL; Cashman, TF; Fraser, ST; New, EJ). Mitochondrially Targeted Redox Probe Reveals the Variations in Oxidative Capacity of the Haematopoietic Cells. Org. Biomol. Chem. 2015, 13, 6686–6690. DOI: 10.1039 / c5ob00928f. Triphenylphosphine (2.62 g, 10 mmol) and 2-bromoethylamine hydrobromide (2.05 g, 10 mmol) were placed in acetonitrile (50 mL) and heated under reflux overnight. After the reaction was complete, the mixture was cooled to room temperature, filtered, and the filter cake was washed three times with cold acetonitrile and dried to give intermediate 4 (3.54 g, yield 75.8%), a white solid.

[0049] 2. Synthesis of intermediate 5: 4-Bromo-1,8-naphthalenedicarboxylic anhydride (3.11 g, 11.2 mmol) and β-alanine (2.0 g, 22.4 mmol) were placed in ethanol (60 mL) and heated under reflux for 6 hours. After the reaction was completed, the mixture was cooled to room temperature, filtered, and the filter cake was washed three times with an ethanol / water mixture (1:1, v / v). After drying, a white solid intermediate 5 (2.83 g, yield 72.4%) was obtained. 1 H NMR (400 MHz, DMSO-) d 6) δ 8.46 (dd, 2H), 8.24 (d, 1H), 8.13 (d, 1H), 7.92 (t, 1H), 4.22 (t, 2H), 2.59 (t, 2H). 13 C NMR (101 MHz, DMSO- d 6 ) δ 172.43, 162.73,162.68, 132.61, 131.53, 131.32, 130.91, 129.72, 129.16, 128.75, 128.20,122.65, 121.87, 35.85, 32.09.HRMS (ESI) m / z: [M + Na] + calcd for C 15 H 10 BrNO4Na,369.9685; found, 369.9684. Intermediate 5 1 The 1H NMR spectrum showed that it had the characteristic proton signal of the naphthalene ring and the methylene signal corresponding to the alanine chain. The molecular ion peak detected by HRMS was consistent with the theoretical calculation value, confirming its structure.

[0050] 3. Synthesis of intermediate 6: Intermediate 5 (1.0 g, 2.9 mmol) was mixed with thionyl chloride (4 mL, 55.1 mmol) and refluxed at 80 °C for 10 hours. Excess thionyl chloride was removed by vacuum distillation, and the residue was redissolved in anhydrous dichloromethane. Anhydrous DMF solution of intermediate 4 (i.e., triphenylphosphine derivative, 1.55 g, 3.3 mmol) and N,N-diisopropylethylamine (DIPEA, 1.2 mL, 6.9 mmol) was added to this solution. The mixture was stirred at room temperature for 4 hours. After solvent removal, the crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 20:1, v / v) to give intermediate 6 (1.34 g, 72.6% yield) as a white solid. 1 H NMR (400 MHz, DMSO- d 6 ) δ 8.51 (dd, 2H), 8.45 (t, 1H), 8.27 (d, 1H),8.19 (d, 1H), 7.97 (t, 1H), 7.92–7.72 (m, 15H), 4.19 (t, 2H), 3.71 (m, 2H),3.30 (m, 2H), 2.39 (t, 2H). 13C NMR (101 MHz, DMSO- d 6 ) δ 170.48, 162.83,135.01, 134.99, 133.64, 133.54, 132.65, 131.50, 131.35, 130.90, 130.28,130.15, 129.80, 129.15, 128.81, 128.30, 122.76, 121.98, 118.60, 117.74,36.46, 33.30, 32.64. HRMS (ESI) m / z: [M] + calcd for C 35 H 29 BrN2O3P, 635.1079; found, 637.1065. Intermediate 6 1 The presence of multiple aromatic proton signals from the triphenylphosphine group in the 1H NMR spectrum confirms the successful introduction of the TPP group.

[0051] 4. Synthesis of intermediate 7: Intermediate 6 (0.5 g, 0.79 mmol) and 1-Boc-piperazine (0.29 g, 1.6 mmol) were dissolved in 2-methoxyethanol (10 mL). The reaction mixture was purged three times with nitrogen and stirred overnight at 130 °C. The solvent was removed under reduced pressure, and the crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 20:1, v / v) to give intermediate 7 (0.45 g, yield 76.8%). 1 H NMR (400 MHz, DMSO- d 6 ) δ 8.54 – 8.42 (m, 2H), 8.40 (d, 1H), 8.31 (d,1H), 7.95 – 7.69 (m, 16H), 4.18 (t, 2H), 3.73 (m, 2H), 3.64 (t, 4H), 3.33 (m,2H), 3.16 (t, 4H), 2.37 (t, 2H), 1.44 (s, 9H). 13C NMR (101 MHz, DMSO) δ170.55, 163.43, 162.91, 155.40, 153.92, 135.01, 134.98, 133.64, 133.54,132.00, 130.59, 130.50, 130.28, 130.15, 129.04, 126.15, 125.37, 122.51,118.61, 117.76, 115.91, 115.39, 79.16, 52.45, 36.17, 33.49, 32.63, 28.06.HRMS (ESI) m / z: [M] + calcd for C 44 H 46 N4O5P, 741.3201; found, 741.3191. 5. Synthesis of intermediate 8: Intermediate 7 (0.4 g, 0.54 mmol) was dissolved in a mixed solution of trifluoroacetic acid (TFA) and dichloromethane (1:1, v / v) and stirred at room temperature for 4 hours. The solution was concentrated under reduced pressure to obtain an oily residue. Upon addition of diethyl ether (5 mL), a yellow solid precipitated. The solid was filtered and washed three times with diethyl ether to obtain intermediate 8 (0.42 g, trifluoroacetate), which was used directly in the next reaction.

[0052] The reaction process in step (2) is as follows: (3) Synthesis of the final product DCINP: 1. Synthesis of DCINP-OH: Intermediate 3 (0.2 g, 0.5 mmol) and intermediate 8 (0.42 g) were dissolved in 1,2-dichloroethane (5 mL), and DIPEA (175 μL, 1 mmol) and sodium cyanoborohydride (0.15 g, 0.7 mmol) were added. The mixture was stirred at room temperature for 1.5 hours. The reaction solution was quenched with saturated sodium bicarbonate solution and extracted with dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 20:1, v / v) to give a purple solid DCINP-OH (0.32 g, yield 62.7%). 1H NMR (500 MHz, CDCl3) δ 8.60 – 8.56 (m, 1H), 8.54(d, 1H), 8.34 (dd, 2H), 7.98 (d, 1H), 7.88 – 7.76 (m, 10H), 7.71 (td, 6H), 7.63 (d, 1H), 7.17 (d, 1H), 6.97 – 6.83 (m, 2H), 6.82 (s, 1H), 4.39 (td, 4H), 3.97 (s, 2H), 3.81 (s, 2H), 3.72 (s, 4H), 3.37 (s, 2H), 3.00 (s, 4H), 2.63 –2.59 (m, 4H), 2.44 (s, 2H), 1.08 (s, 6H). 13 C NMR (126 MHz, CDCl3) δ 172.04,171.89, 169.17, 163.39, 153.80, 135.31, 135.28, 133.68, 133.60, 133.17,131.92, 131.11, 130.99, 130.62, 130.52, 130.12, 129.92, 129.08, 127.98,126.22, 123.18, 118.31, 117.62, 42.99, 39.22, 36.70, 35.92, 33.83, 33.04,32.04, 31.91, 29.78, 29.70, 29.62, 29.53, 29.36, 29.32, 29.26, 28.03, 27.22,25.56, 22.69, 14.13. HRMS (ESI) m / z: [M] + calcd for C 59 H 55 BrN6O4P, 1023.3196;found, 1023.3228. 2. Synthesis of the final product DCINP: DCINP-OH (0.1 g, 0.1 mmol) was dissolved in dichloromethane, followed by the addition of acryloyl chloride (12 μL, 0.15 mmol) and DIPEA (26 μL, 0.15 mmol). The mixture was stirred at room temperature for 6 hours. After the reaction was complete, the solvent was removed under reduced pressure, and the crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 20:1, v / v) to give the yellow solid final product probe DCINP (68.5 mg, yield 63.6%).1 H NMR (500 MHz, CDCl3) δ 8.47 (dd, 2H), 8.24(d, 2H), 7.90 (d, 1H), 7.77 – 7.69 (m, 10H), 7.62 (dt, 6H), 7.53 (t, 1H), 7.03 (d, 1H), 6.97 (s, 2H), 6.81 (s, 1H), 6.50 (dd, 1H), 6.12 (dd, 1H), 6.01(dd, 1H), 4.32 (t, 2H), 3.85 – 3.69 (m, 6H), 3.63 (m, 6H), 3.31 (q, 2H), 2.54(d, 4H), 2.40 (s, 2H), 1.00 (s, 6H). 13 C NMR (126 MHz, CDCl3-d) δ 171.08,168.20, 162.56, 160.25, 147.09, 134.21, 133.64, 132.68, 132.59, 132.21,130.89, 129.95, 129.82, 129.57, 129.46, 128.85, 126.95, 126.89, 126.48,125.45, 122.15, 121.30, 117.33, 116.64, 42.03, 38.18, 35.72, 32.87, 32.02,31.03, 29.38, 28.53, 28.68, 28.30, 26.96, 26.19, 23.66, 21.64, 13.10. HRMS(ESI) m / z: [M] + calcd for C 62 H 57 BrN6O5P, 1077.3303; found, 1077.3326. High-resolution mass spectrometry (HRMS) analysis results from DCINP showed that its [M] + The value is 1077.3326, which is consistent with the molecular formula C of DCINP. 62 H 57 The theoretical value of BrN6O5P is consistent with 1077.3303.

[0053] The reaction process in step (3) is as follows: The above analysis data shows that the target probe DCINP and all its key intermediates were successfully synthesized and have the correct structure.

[0054] Figure 1 This diagram illustrates the design principle and optical performance characterization of the probe DCINP, and includes three sub-diagrams. Figure 1 The diagram in (a) is a schematic diagram of the reaction mechanism between the probe DCINP and cysteine ​​(Cys). This diagram was drawn based on the molecular design principle of the probe and the verified reaction mechanism, and aims to intuitively demonstrate its synergistic sensing mechanism based on ICT-FRET. Figure 1 (b) shows the spectral overlap of the donor (NP) and acceptor (DCI) units to demonstrate that they meet the conditions for fluorescence resonance energy transfer (FRET). The energy donor unit (naphthalimide-piperazine, NP) and the energy acceptor unit (cyanoisophorone derivative, DCI) were synthesized and purified, respectively. The absorption spectra of NP and DCI were scanned using a UV-Vis spectrophotometer, and the fluorescence emission spectra of both were acquired using a fluorescence spectrometer. The normalized fluorescence emission spectrum of the obtained NP and the normalized UV absorption spectrum of DCI were plotted on the same coordinate system to show their spectral overlap. Figure 1 (c) shows a comparison of the fluorescence spectra of the system before and after the reaction, demonstrating the ratiometric fluorescence response induced by the reaction of probe DCINP with Cys. Four sets of test samples were set up, including NP solution (donor control), DCINP solution (free probe), DCINP-OH solution (reaction product control), and DCINP+Cys. The fluorescence emission spectra of the above samples were collected using a fluorescence spectrometer, and the four sets of spectra were plotted in the same figure. Figure 1 (c) shows a significant shift in the fluorescence peak from 540 nm (green, donor emission) to 660 nm (near-infrared red, acceptor emission) before and after the reaction, as well as the high consistency between the spectra of DCINP-OH and the "DCINP+Cys" reaction system to verify the reaction mechanism.

[0055] Example 2 Spectral performance testing of probe DCINP: Prepare a 5 μM DCINP stock solution (DMSO as solvent). All spectral assays were performed in PBS / ethanol (1:1, v / v, pH 7.4) buffer at room temperature for 30 minutes.

[0056] Selectivity experiment: 50 μM of various potential interfering substances (including anions, cations, glucose, various amino acids, Hcy, GSH) or 25 μM of Cys were added to DCINP solution, and the fluorescence intensity I at 540 nm and 660 nm was measured. 540 and I 660 And calculate I 660 / I 540 The ratio, the result is as follows Figure 2 As shown.

[0057] Figure 2 The display shows that I only exists in the presence of Cys. 660 / I 540 The ratio increased sharply, while other substances had no significant effect.

[0058] Sensitivity and linearity: Different concentrations of Cys (2, 5, 8, 10, 12.5, 15, 17.5, 20, 25, 30, 35, 40, 45, 50, 55, and 60 μM) were added to DCINP solution. Fluorescence intensity curves were measured at each concentration. 540 and I 660 The result is as follows Figure 3 As shown, I was calculated based on the obtained fluorescence intensity curve. 660 / I 540 The ratio was examined, and its linear relationship was observed. The results are as follows: Figure 4 As shown.

[0059] Figures 3~4 The results showed that after adding Cys, the fluorescence emission peak shifted from 540 nm to 660 nm. 660 / I 540 The ratio showed a good linear relationship with Cys concentration in the range of 0–40 μM, and the linear equation was I. 660 / I 540 =0.5807[Cys]+0.8913(R 2 =0.9881), and the detection limit is calculated to be 37 nM.

[0060] pH stability: 25 μM Cys was added to the DCINP solution, and the pH was adjusted to 5.0, 6.0, 6.5, 7.0, 7.4, 8.0, 8.5, and 9.0. The free probe was tested within the pH range of 5.0–9.0. 660 / I 540 Stability of the ratio: Test results show that the ratio after reaction with Cys reaches its maximum and remains stable at pH 6.0~9.0, especially at physiological pH 7.4, indicating that the probe DCINP prepared in this invention is suitable for physiological environments.

[0061] Example 3 Cytotoxicity: The cytotoxicity of the probe DCINP against HepG2 cells was evaluated using the CCK-8 assay. The specific steps were as follows: HepG2 cells in logarithmic growth phase were cultured at 8 × 10⁸ cells per well. 3Cells were seeded at a density of 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in DMEM medium and incubated at 37°C, 5% CO2 for 24 hours to allow adherence. The original medium was discarded, and fresh medium containing different concentrations of DCINP (0–40 μM) was added, followed by incubation for another 24 hours. Subsequently, the drug-containing medium was discarded, and the cells were gently washed three times with PBS. 100 μL of a fresh mixture of CCK-8 reagent and DMEM at a 1:9 (v / v) ratio was added to each well, and incubation continued for 6 hours. Finally, the absorbance of each well was measured at 450 nm using a microplate reader. Cell viability (%) was calculated using the following formula: Cell viability (%) = [(OD...] 450 Sample – OD 450 Blank) / (OD 450 Comparison – OD 450 [Blank)]×100%.

[0062] Experimental results showed that even with a DCINP concentration as high as 40 μM, the survival rate of HepG2 cells was still >80%, indicating that it has low cytotoxicity.

[0063] Mitochondrial colocalization: To verify the mitochondrial targeting ability of the probe, a co-localization experiment was performed. Cultured HepG2 cells were pretreated with N-ethylmaleimide (NEM) for 30 minutes to consume endogenous thiols. Then, they were co-incubated with DCINP and the commercially available mitochondrial dye MitoTracker Red for 60 minutes. After washing with PBS, imaging was performed using a confocal laser scanning microscope. DCINP fluorescence was excited using a 488 nm laser, and the emission signal was collected in the green channel; MitoTracker Red fluorescence was excited using a 579 nm laser, and the emission signal was collected in the red channel. The confocal imaging results are shown below. Figure 5 (The scale bar in the figure is 25 μm), where a is the green channel fluorescence image of DCINP, b is the red channel fluorescence image of MitoTracker Red, c is the bright field image, d is the overlay of a, b, and c, e is the co-localization scatter plot of DCINP and MitoTracker Red fluorescence, and f is the fluorescence intensity distribution curve along the selected white arrow direction.

[0064] Figure 5 The results showed that the green channel fluorescence of DCINP highly overlapped with the red channel fluorescence of MitoTracker Red, with a Pearson correlation coefficient of 0.89, demonstrating its excellent mitochondrial targeting.

[0065] Intracellular Cys ratio imaging: To evaluate the probe's specific ratioographic imaging capability for Cys in live cells, HepG2 cells were divided into different treatment groups: Group 1 was incubated with 20 μM DCINP for 60 min; Group 2 was pretreated with 20 μM exogenous Cys for 30 min, then co-incubated with 20 μM M DCINP for 60 min; Group 3 was pretreated with 100 μM NEM for 45 min, then incubated with 20 μM DCINP for 60 min; Groups 4-6 were pretreated with 100 μM NEM for 45 min, then co-incubated with 200 μM GSH, Hcy, or Cys for 30 min respectively, and finally incubated with 20 μM DCINP for 60 min. All groups were washed three times with PBS after treatment and imaged using confocal microscopy (λ). ex =488 nm; Green channel λ em 500~600 nm, red channel λ em (600~700 nm). The ratio fluorescence imaging results (a) and quantitative analysis results (b) of the probe DCINP in the mitochondria of live cells (HepG2) are shown below. Figure 6 .

[0066] Figure 6 The results showed that cells only exhibited strong red channel fluorescence and a high red / green fluorescence intensity ratio (IL) in the presence of endogenous or exogenous Cys. Red / I Green Quantitative analysis confirmed that the probe could specifically distinguish Cys from Hcy / GSH and successfully monitor changes in intracellular Cys levels.

[0067] Example 4 In vivo imaging applications of the DCINP probe: Prior to imaging, mice underwent hair removal on their abdomens and were under general anesthesia using isoflurane inhalation. To eliminate interference from endogenous thiols, NEM solution (1 mM) was first injected subcutaneously into both left and right inguinal regions of the mice to consume endogenous thiols. Subsequently, probe solution (5 mM) was injected into the same locations on both sides. Finally, exogenous Cys solution (5 mM) was injected only on the right side to induce a specific response, with the left side serving as a control. Monitoring was performed using a small animal in vivo imaging system (IVIS Lumina Series III), and fluorescence images of the green and red channels were acquired at 0, 30, and 60 minutes after injection. The ratio fluorescence imaging results of probe DCINP against exogenous Cys in vivo in mice (a) and the red / green fluorescence intensity ratio (b) are shown in the figure. Figure 7 .

[0068] Figure 7 The results showed that, over time, the fluorescence in the red channel on the side injected with Cys significantly increased, and the red / green fluorescence intensity ratio (IL) increased. Red / I Green The signal on the control side continued to rise, while the signal on the control side remained stable at a low level. This demonstrates that DCINP can achieve ratio fluorescence imaging of Cys at the in vivo level.

[0069] Example 5 Application of DCINP probe in food testing and construction of smartphone platform: Sample pretreatment: Take garlic, dried red pepper, tomato, onion, carrot, and apple, homogenize them separately, and then extract them by sonication with DMSO / PBS (1:1, V / V) solution. Centrifuge and collect the supernatant (see process). Figure 8 (a)

[0070] Fluorescence detection and spiked recovery: The processed sample extract was added to the DCINP probe and detected. The standard addition method was used for spiked recovery experiments, and the results are shown in Table 1. Table 1 shows that the recovery rates of each sample ranged from 92.10% to 114.73%, with an RSD of less than 7%, indicating that the method is accurate and reliable, and the DCINP probe can be used to detect Cys in food.

[0071] Table 1 Portable smartphone detection platform: Solutions containing different concentrations of Cys standards or food extracts reacting with DCINP are placed under a UV lamp (365 nm). A smartphone is used to take a picture, and image processing software (such as ColorPicker or ImageJ) is used to extract the RGB values ​​of the reaction area in the image. A standard curve is established with the red / green channel intensity ratio (R / G) as the ordinate and the Cys concentration as the abscissa (see [link to standard curve]). Figure 8 (b) R / G = 0.1235C + 0.6808 (R 2 =0.9915), LOD is 0.065 μM. This standard curve can be used for rapid quantification of unknown food samples, and the results are highly consistent with those of HPLC, enabling low-cost, portable, on-site rapid visual-quantitative dual-mode detection.

[0072] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A ratiometric near-infrared fluorescent probe based on the ICT-FRET mechanism, characterized in that, The structural formula is shown below: 。 2. A method for preparing the ratiometric near-infrared fluorescent probe based on the ICT-FRET mechanism as described in claim 1, characterized in that, Includes the following steps: Starting with 4-bromo-1,8-naphthalenedicarboxylic anhydride and β-alanine, a carboxylic acid intermediate was generated through a condensation reaction. The carboxylic acid intermediate was then reacted with thionyl chloride to generate an acyl chloride intermediate. The acyl chloride intermediate was then reacted with a triphenylphosphine derivative via an amidation reaction to obtain a triphenylphosphine-modified naphthalenedicarboxylic anhydride intermediate. The triphenylphosphine-modified naphthalenedicarboxylic anhydride intermediate was then subjected to a nucleophilic substitution reaction with 1-Boc-piperazine, followed by removal of the Boc protecting group and introduction of a piperazine linker to obtain an amino intermediate. The amino intermediate and the cyanoisophorone derivative were covalently linked by a reductive amination reaction to obtain the precursor compound; the precursor compound was reacted with acryloyl chloride under alkaline conditions to obtain the ratiometric near-infrared fluorescent probe based on the ICT-FRET mechanism. The structural formula of the carboxylic acid intermediate is as follows: ； The structural formula of the triphenylphosphine derivative is: ； The structural formula of the triphenylphosphine-modified naphthalenedicarboximide intermediate is as follows: ； The structural formula of the amine intermediate is: ； The structural formula of the cyanoisophorone derivative is: ； The structural formula of the precursor compound is: 。 3. The use of the ratiometric near-infrared fluorescent probe based on the ICT-FRET mechanism as described in claim 1 in the preparation of reagents for fluorescent imaging of cysteine ​​in mitochondria of cells or living animals.

4. The use of the ratiometric near-infrared fluorescent probe based on the ICT-FRET mechanism as described in claim 1 in the preparation of reagents or kits for the detection of cysteine ​​in food.

5. The application of the ratiometric near-infrared fluorescent probe based on the ICT-FRET mechanism as described in claim 1 in the preparation of a cysteine ​​detection kit.

6. A method for quantitative detection of cysteine ​​based on an RGB analysis system, characterized in that, Includes the following steps: The ratiometric near-infrared fluorescent probe based on the ICT-FRET mechanism described in claim 1 was mixed with different concentrations of cysteine ​​and photographed under a UV lamp. The RGB values ​​of the photographs were extracted using an RGB analysis system. A standard curve was established using the red / green channel intensity ratio of the obtained RGB values ​​and the corresponding cysteine ​​concentration as parameters. The sample to be tested was mixed with the ratiometric near-infrared fluorescent probe based on the ICT-FRET mechanism described in claim 1 and photographed under a UV lamp. The RGB values ​​of the photograph were extracted using an RGB analysis system. The cysteine ​​content of the sample to be tested was calculated based on the standard curve.

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