A near-infrared two-photon dual-mode linked response NTR & Cys fluorescent probe, its preparation method and application

Abstract

This invention discloses a near-infrared two-photon dual-mode linked response NTR&Cys fluorescent probe, its preparation method, and its application, belonging to the field of medical detection technology. The structural formula of the near-infrared two-photon dual-mode linked response NTR&Cys fluorescent probe is as follows: The near-infrared two-photon dual-mode linked response NTR&Cys fluorescent probe of this invention possesses a hypoxia dual-mode visual monitoring molecular framework, enabling dual-mode determination of hypoxia microenvironments. It improves the stability of the cyanine near-infrared framework and achieves highly stable and specific detection of nitroreductase under hypoxia. Under hypoxia conditions, the product reacts with NTR, and the single-photon excited near-infrared fluorescence is quenched and then illuminated. Simultaneously, under hypoxia, the probe reacts with Cys to generate a two-photon excited fluorescence signal, effectively avoiding the probe consumption problem of dual-response systems and ensuring the non-interference of fluorescence excitation and emission (dual-mode), thus truly achieving accurate locking of hypoxia microenvironments.

Description

Technical Field

[0001] This invention belongs to the field of medical detection technology, specifically relating to a near-infrared two-photon dual-mode linkage response NTR&Cys fluorescent probe, its preparation method, and its application. Background Technology

[0002] Nitroreductase (NTR), an endogenous enzyme highly expressed in hypoxic tumor cells, is closely related to the degree of hypoxia in tumor tissue. Nitroreductase is an indicative biomarker of tumors and cancer complications, and is therefore used as a basic diagnostic and therapeutic target for cancer. Cysteine ​​(Cys) is one of the essential amino acids in the human body, playing a crucial role in maintaining biological redox homeostasis. Simultaneously, Cys exhibits a high level of distribution in tumor tissues, especially under hypoxic conditions, and can also serve as a contributing factor in detecting and indicating the tumor microenvironment.

[0003] Near-infrared (NIR) and two-photon (2PS) probes are widely used in the analysis of various disease biomarkers due to their unique optical properties. Based on numerous strategies, researchers have developed many NIR or 2PS fluorescent probes for monitoring NTR and Cys. However, these probes only respond independently to NTR and Cys, and almost all of them emphasize NTR as a biomarker of tumor hypoxia. This approach is too simplistic in evaluating the degree of tumor hypoxia and is prone to unavoidable false positives due to various factors, increasing the uncertainty of the detection results. Furthermore, currently reported dual-response probes do not possess dual-mode detection capabilities for both NIR and 2PS, leaving room for further research and opportunities in the field of probe development.

[0004] Therefore, the synergistic detection of NTR and Cys is of great significance for accurately identifying the tumor microenvironment, avoiding cancer detection errors, and precision medicine. Summary of the Invention

[0005] To address the problems and deficiencies in existing technologies, this invention provides a near-infrared two-photon dual-mode linkage response NTR & Cys fluorescent probe, its preparation method, and its application. This enables interference-free continuous monitoring of NTR and Cys under hypoxic conditions using single-molecule near-infrared and two-photon dual-channel methods, making the localization of the cancer microenvironment more accurate.

[0006] This invention is achieved through the following technical solution:

[0007] A near-infrared two-photon dual-mode linked response NTR & Cys fluorescent probe, the structure of which is shown below:

[0008]

[0009] In this invention, the preparation method of the above-mentioned near-infrared two-photon dual-mode linked response NTR&Cys fluorescent probe includes the following steps:

[0010] (1) Anhydrous potassium carbonate was dissolved in water, deoxygenated and recrystallized, and transferred to a deoxygenated acetone solution of 4-(4-hydroxyphenyl)cyclohexanone and p-nitrobenzyl bromide. The mixture was heated to reflux. After the reaction was completed, the solution was cooled to room temperature and filtered. The mother liquor was treated with activated carbon and concentrated at low temperature in the dark to obtain a yellow solid crude product. The crude product was recrystallized with petroleum ether and purified to obtain compound 2.

[0011] (2) Freshly distilled DMF and CH2Cl2 were placed in a cold trap and stirred. Then, they were added to a mixture of CH2Cl2 and POCl3 and gradually raised to room temperature. Then, they were added dropwise to compound 2. The resulting mixture was heated and refluxed. After the reaction was completed, the reaction solution was placed in ice water overnight in the dark. It was extracted with a mixture of ethyl acetate-methanol-dichloromethane, dried with anhydrous Na2SO4, and vacuum dried to obtain compound 3.

[0012] (3) Mix compound 3 with sodium acetate and add acetic anhydride and stir thoroughly to obtain a mixture. Add acetic anhydride solution of compound 1 dropwise to the mixture. Place the resulting mixture under light-protected conditions and increase the temperature gradually. After the reaction is completed, concentrate under reduced pressure and wash with a mixture of dichloromethane and methanol to obtain compound 4.

[0013] (4) Under helium protection, 6-hydroxy-2-naphthaldehyde and NaH were dissolved in freshly distilled DMF and stirred. The DMF solution of compound 4 was slowly added and the reaction was carried out at room temperature in the dark. After the reaction was completed, the reaction solution was extracted with a mixture of dichloromethane and ethyl acetate in the dark. The extract was placed at low temperature and allowed to stand. The organic layer was dried with anhydrous Na2SO4 and the solvent was removed under vacuum to obtain the crude product. After purification by silica gel column, the near-infrared two-photon two-mode linkage response NTR&Cys fluorescent probe was obtained.

[0014] The reaction formula for preparing the near-infrared two-photon dual-mode linked response NTR & Cys fluorescent probe is as follows:

[0015]

[0016] Further, in step (1), the molar ratio of 4-(4-hydroxyphenyl)cyclohexanone to 4-nitrobenzyl bromide is 3:4; in step (2), the ratio of POCl3 to compound 2 is 1 mL: 1 g; in step (3), the molar ratio of compound 3 to compound 1 is 1:2; in step (4), the molar ratio of 6-hydroxy-2-naphthaldehyde, NaH to compound 4 is 5:5:1.

[0017] Further, in step (2), the volume ratio of DMF, CH2Cl2 and POCl3 is 1:2:1; in step (3), the mass ratio of compound 3 to sodium acetate and acetic anhydride in the mixture is 1:1:1, and the mass ratio of compound 1 to acetic anhydride in the acetic anhydride solution of compound 1 is 3.2:1.

[0018] Furthermore, in step (1), the reaction temperature of heating and reflux is 60°C and the reaction time is 13h; in step (3), the gradient heating condition is 4h to 70°C; in step (4), the reaction time at room temperature in the dark is 6h.

[0019] Furthermore, the temperature of the cold trap or cryogenic environment is -5 to -10°C.

[0020] Further, in step (2), the volume ratio of ethyl acetate, methanol, and dichloromethane in the ethyl acetate-methanol-dichloromethane mixture is 1:8:1; in step (3), the volume ratio of dichloromethane to methanol in the dichloromethane-methanol mixture is 10:1; and in step (4), the volume ratio of dichloromethane to ethyl acetate in the dichloromethane-ethyl acetate mixture is 4:1.

[0021] In this invention, the near-infrared two-photon dual-mode linkage response NTR&Cys fluorescent probe is used in the preparation of tumor detection reagents.

[0022] Cyanide near-infrared molecular frameworks are highly susceptible to photodegradation, which significantly limits their applications. This invention addresses this issue by modifying functional groups in the strong ultraviolet region that causes decomposition. Benzene and benzyl nitro groups are introduced into the lower part of the cyanide framework, fully utilizing the single-molecule internal filtering effect to achieve absorption in the ultraviolet region, reducing the decomposition of the cyanide framework and effectively improving its stability. The tail-end nitrobenzyl structure can sensitively trigger an anaerobic environment, and the introduction of a naphthalyl group into the middle position of the cyanide framework constructs an interference-free two-photon excitation mode fluorescence signal. A naphthalene ring as a linking group between the near-infrared fluorescent framework and the Cys triggering structure ensures synergistic and interference-free analysis between two-photon and near-infrared excitation; simultaneously, a non-conjugated structure connects to the cyanide framework, eliminating the fluorescence background of the cyanide framework through photoinduced electron transfer. Furthermore, the nitro group of the p-nitrobenzyl group specifically responds to nitroreductase, thereby preventing photoinduced electron transfer and causing the near-infrared framework to release fluorescence. Therefore, this molecule optimization enhances the stability of the cyanide near-infrared framework, achieving highly stable and specific detection of nitroreductase.

[0023] First, by attaching a benzene ring below the near-infrared cyanine structure, this invention achieves an enhanced internal filtration effect, thus avoiding the decomposition effect of low-wavelength light on the product. Simultaneously, this group can sensitively respond to hypoxic environments. Second, the naphthaldehyde group structure assembled at the meso-position of the cyanine can form a certain photoinduced electron transfer effect and construct a two-photon active center, responding to Cys molecules under hypoxic conditions, thereby generating enhanced detection. Third, based on the design and coordination of the above molecular components, the product can achieve interference-free dual-mode visual monitoring of two factors under cellular hypoxia. Specifically, under the action of the hypoxia marker nitroreductase, near-infrared fluorescence is initially triggered from a closed state; as the degree of hypoxia intensifies, the fluorescence gradually increases; simultaneously, Cys under hypoxia triggers the two-photon active group, generating a two-photon signal. The intersection region of the signals from the two modes is the precise hypoxic region, thereby improving the fidelity of tumor microenvironment hypoxia detection.

[0024] Beneficial effects

[0025] The near-infrared two-photon dual-mode linked response NTR & Cys fluorescent probe of this invention possesses a novel molecular framework for hypoxia dual-mode visual monitoring. It synthesizes a molecular probe capable of dual-mode determination of hypoxic microenvironments, improves the stability of the cyanine near-infrared framework, and achieves highly stable and specific detection of nitroreductase under hypoxia. Under hypoxic conditions, the product reacts with NTR, and the single-photon excited near-infrared fluorescence is quenched and then illuminated; simultaneously, under hypoxic conditions, the probe reacts with Cys to generate a two-photon excited fluorescence signal. Furthermore, it effectively avoids the probe consumption problem of dual-response systems and ensures interference-free fluorescence excitation and emission (dual-mode), thus truly achieving accurate targeting of hypoxic microenvironments. Attached Figure Description

[0026] Figure 1 The nuclear magnetic resonance (HNMR) spectrum of compound 2 prepared in Example 1;

[0027] Figure 2 The nuclear magnetic resonance (HNMR) spectrum of compound 4 prepared in Example 1;

[0028] Figure 3 The near-infrared two-photon dual-mode linkage response NTR & Cys fluorescent probe prepared in Example 1 is shown in the nuclear magnetic resonance (HNMR) spectrum.

[0029] Figure 4 The CNMR spectrum of the near-infrared two-photon dual-mode linked response NTR & Cys fluorescent probe prepared in Example 1;

[0030] Figure 5 Fluorescence spectra and linear relationships of near-infrared two-photon two-mode linkage response NTR & Cys fluorescent probes for NTR and Cys;

[0031] Figure 6 The kinetic curves of the near-infrared two-photon two-mode linkage response NTR & Cys fluorescent probe to NTR are shown.

[0032] Figure 7 The kinetic curves of the near-infrared two-photon two-mode linkage response NTR & Cys fluorescent probe to Cys response;

[0033] Figure 8 The figure shows the selectivity test results of the near-infrared two-photon two-mode linkage response NTR & Cys fluorescent probe;

[0034] Figure 9 The mass spectrometry monitoring diagram of the near-infrared two-photon dual-mode linkage response NTR & Cys fluorescent probe and the NTR and Cys response;

[0035] Figure 10 Schematic diagram of near-infrared two-photon dual-mode linkage response NTR & Cys fluorescent probes and intermediate cytotoxicity;

[0036] Figure 11 This is a dual-mode fluorescence imaging image of the near-infrared two-photon dual-mode linkage response NTR & Cys fluorescent probe in cells;

[0037] Figure 12 This is a dual-mode fluorescence imaging image of a near-infrared two-photon dual-mode linkage response NTR & Cys fluorescent probe in mouse liver tissue. Detailed Implementation

[0038] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods.

[0039] In the following examples, the near-infrared two-photon dual-mode linkage response NTR&Cys fluorescent probe (OTA) was dissolved in PBS buffer solution (pH=7.4, 1% DMSO, 0.01M).

[0040] Example 1

[0041] The preparation of a near-infrared two-photon two-mode linked response NTR & Cys fluorescent probe is shown in the following reaction formula:

[0042]

[0043] Specifically:

[0044] (1) Preparation of compound 2 (DOB): 8.36 g (0.06 mol) of anhydrous potassium carbonate was dissolved in 30 mL of freshly prepared deionized water, deoxygenated under nitrogen for 30 minutes, and then recrystallized. The entire solution was transferred to a solution of 5.7 g (0.030 mol) of 4-(4-hydroxyphenyl)cyclohexanone and 8.64 g (0.04 mol) of 4-nitrobenzyl bromide dissolved in 120 mL of deoxygenated acetone. The resulting mixture was refluxed at 60 °C for 13 h. After the reaction, the solution was cooled to room temperature and filtered. The mother liquor was treated with activated carbon (20 g) and concentrated at 20 °C in the dark to obtain a yellow crude solid. The crude solid was then recrystallized from petroleum ether and purified to obtain 8.3 g (85% yield) of pure compound 2. The nuclear magnetic resonance (NMR) spectrum of compound 2 is shown below. Figure 1 As shown;

[0045] (2) Preparation of compound 2: DMF (5 mL) after dehydration and deoxygenation and CH2Cl2 (5 mL) were placed in a flask and stirred for 30 min at -10 °C. The resulting solution was added to a mixture of 5 mL CH2Cl2 and 4 mL POCl3 and gradually raised to room temperature (25 °C). Then it was added dropwise to a flask containing compound 2 (4 g, 0.012 mol). The resulting mixture was rapidly heated under reflux and the reaction was monitored until complete. The resulting solution was poured into a beaker containing ice and kept in the dark overnight (8 h). Finally, it was extracted with a mixture of ethyl acetate / methanol / dichloromethane (v:v:v = 1:8:1), dried with anhydrous Na2SO4 and vacuum evaporated to obtain compound 3.

[0046] (3) Preparation of compound 4: Compound 3 (2g, 0.005mol) was mixed with sodium acetate (2g, 0.005mol) and acetic anhydride (2g, 0.005mol) was added and stirred thoroughly (1h). Then, a solution of acetic anhydride (1g, 0.0025mol) of compound 1 (3.2g, 0.01mol) was added. The resulting mixture was placed under light-protected conditions and heated gradually (70℃ / 4h) for 11 hours. After the reaction, the mixture was concentrated under reduced pressure and washed with a mixture of dichloromethane and methanol (CH2Cl2 / CH3OH = 10:1) to obtain the green solid product compound 4. The nuclear magnetic resonance (NMR) spectrum of compound 4 is shown below. Figure 2 As shown;

[0047] (4) Synthesis of near-infrared two-photon two-mode linkage response NTR&Cys fluorescent probe (OTA): Under helium protection, 6-hydroxy-2-naphthaldehyde (0.43 g, 0.0025 mol) and NaH (0.1 g, 0.0025 mol) (containing 40% mineral oil) were dissolved in freshly distilled DMF in a flask and stirred at room temperature for 30 minutes. Then, compound 4 (0.433 g, 0.0005 mol) was dissolved in 10 mL of freshly distilled DMF and slowly injected into the flask using a syringe. The reaction was carried out at room temperature in the dark for 6 hours. Extraction was performed with a dichloromethane-ethyl acetate mixture (4:1) under dark conditions, followed by standing at 5°C for 3 hours. The organic layer was dried with anhydrous Na2SO4 and the solvent was removed under vacuum to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain a green near-infrared two-photon two-mode linkage response NTR&Cys fluorescent probe (OTA). Its nuclear magnetic resonance (NMR) and nuclear magnetic resonance (NMR) spectra are shown below. Figure 3 and Figure 4 As shown.

[0048] Example 2

[0049] The feasibility of the near-infrared two-photon dual-mode linked response NTR & Cys fluorescent probe (OTA) prepared in Example 1 with NTR and Cys reactions was studied, specifically as follows:

[0050] NTR response: A 500 μM NADH solution was prepared, and the probe OTA (10 μM) was added to it. Then, different concentrations (0-5 μg / mL) of NTR were added, and their fluorescence spectra are shown below. Figure 5 As shown in (a), the fluorescence intensity at 865 nm increases significantly with increasing NTR concentration, and exhibits a good linear relationship with a linear correlation coefficient of 0.9794.

[0051] NTR & Cys linkage response: First, in the presence of 500 μM NADH, compound OTA (10 μM) was fully reacted with 5 μg / mL NTR for 3 h. Then, different concentrations (0-45 μM) of Cys were added, and the fluorescence spectra were as follows. Figure 5 As shown in (b), the fluorescence intensity at around 500 nm increases significantly with the increase of Cys concentration, and shows a good linear relationship with the concentration of Cys with a linear correlation coefficient of 0.9792.

[0052] Example 3

[0053] The kinetic properties of the near-infrared two-photon dual-mode linked response NTR&Cys fluorescent probe (OTA) prepared in Example 1 were tested, specifically as follows:

[0054] To investigate the response of OTA to NTR, NTR was added to the probe OTA (10 μM) in the presence of 500 μM NADH to achieve a concentration of 5 μg / mL. Fluorescence intensity was measured at each time point from 0 min to 90 min after NTR addition, and kinetic curves (Ex = 560 nm, Em = 865 nm) were plotted. The results are as follows: Figure 6 As shown; by Figure 6 As can be seen, the system without NADH showed no obvious signal, while the signal of this system increased over time and remained stable after about 60 minutes.

[0055] To investigate the time-dependent response of OTA to Cys, in the presence of 500 μM NADH, the probe OTA (10 μM) was first subjected to complete NTR treatment, followed by the addition of 45 μM Cys. Fluorescence intensity was measured and recorded at each time point from 0 min to 60 min, and kinetic curves were plotted (Ex = 420 nm, Em = 500 nm). The results are as follows: Figure 7 As shown; by Figure 7 As shown, as the interaction time between Cys and the probe increases, the fluorescence intensity of the probe gradually increases, and the fluorescence intensity tends to stabilize at about 40 minutes.

[0056] Example 4

[0057] The response selectivity of the near-infrared two-photon dual-mode linked response NTR&Cys fluorescent probe (OTA) prepared in Example 1 was tested, specifically as follows:

[0058] In the presence of NADH (500 μM), the probe OTA (10 μM) was reacted with various interfering agents (1.K). + (1mM), 2.Mg 2+ (1mM), 3.Ca 2+ (500μM), 4.F - (500μM), 5.Hg 2+ (500μM), 6.ClO - (500μM), 7.0 2- (500μM), 8.H2O2 (500μM), 9.Gly (500μM), 10.NO 3- (500μM), 11.NO (500μM), 12.HCY (500μM), 13.GSH (500μM), 14.Fe 2+ (500μM), 15.DTT (500μM), 16.NTR (5μg / mL), results (Ex = 560nm, Em = 865nm) Figure 8As shown in (a), the probe OTA exhibits a significant fluorescence change only in the presence of NTR. Other ultra-high concentrations of simulated interfering species have very little effect on the fluorescence intensity of the probe OTA.

[0059] Then, the probe OTA (10 μM) was fully reacted with NTR before OTB reacted with various interfering species (1.HSO3). - (50μM), 2.S2O3 2- (50μM), 3.HS - (50μM), 4.S 2- (50μM), 5.Val(50μM), 6.Gln(50μM), 7.His(50μM), 8.Ser(50μM), 9.Pro(50μM), 10.Gly(50μM), 11.Lys(50μ M), 12.Leu(50μM), 13.Met(50μM), 14.Tyr(50μM), 15.Asp(50μM), 16.Cys(45μM)), the results (Ex=420nm, Em=500nm) are as follows Figure 8 As shown in (b); by Figure 8 (b) It can be seen that obvious fluorescence intensity is only observed in the presence of Cys, and other high concentrations of interfering species cannot cause significant changes in fluorescence.

[0060] Therefore, probes OTA and OTB can continuously and selectively recognize NTR and Cys in complex biological environments, making them a reliable molecular detection tool.

[0061] Example 5

[0062] The response mechanism of the near-infrared two-photon dual-mode linked response NTR&Cys fluorescent probe (OTA) prepared in Example 1 was analyzed, specifically as follows:

[0063] The reaction solutions of probe OTA and NTR, and the reaction solutions of probe OTA and Cys and NTR after successive reactions, were taken separately. The products of the reaction were characterized by high-resolution mass spectrometry. The results are as follows: Figure 9 As shown in (a) and 9(b); by Figure 9 (a) It can be seen that after the probe OTA reacts with NTR, the mass spectrum mainly shows an ion peak at m / z = 844, indicating that in the presence of 500 μM NADH, the nitro moiety of the probe OTA is reduced to an amino group by NTR. The new ion peak is the ion peak OTB of the intermediate formed after the probe OTA combines with NTR. Figure 9(b) It can be seen that when Cys is added to the reaction solution of OTA and NTR, a new ion peak of m / z = 947 is clearly observed in the mass spectrum, indicating that this ion peak is the ion peak of the new product OTC generated after the intermediate cyclizes with Cys. The above high-resolution mass spectrometry experiment verifies the mechanism of continuous interaction between the probe and NTR and Cys.

[0064] Example 6

[0065] The cytotoxicity of the near-infrared two-photon dual-mode linked response NTR&Cys fluorescent probe (OTA) and intermediate OTB prepared in Example 1 was tested, specifically as follows:

[0066] The MTT assay was used to study the cytotoxicity of OTA and its intermediate OTB (the intermediate product of the reaction between OTA and NTR) probes on cells. The results are as follows: Figure 10 As shown; by Figure 10 It can be seen that when the concentration of probes OTA and OTB reaches 100 μM, the survival rate of HeLa is still not less than 70%, which proves that the probe has good biocompatibility with cells and has the potential to be applied to live cell level analysis.

[0067] The near-infrared two-photon dual-mode linked response NTR&Cys fluorescent probe (OTA) prepared in Example 1 was subjected to dual-pathway fluorescence imaging analysis of cells, specifically as follows:

[0068] Imaging of NTR and Cys with OTA (10 μM) in single-photon and two-photon excitation modes, respectively, results are as follows: Figure 11 As shown, where, Figure 11 (a) HeLa cells were cultured for 12 h under different oxygen concentrations (20% O2, 5% O2, 1% O2), and then incubated with the probe OTA (10 μM) for 60 min. Figure 11 (b) Fluorescence images after HeLa cells were cultured with probe OTA (10 μM) for 12 h under hypoxic (1% O2) conditions and then incubated with different concentrations of Cys (0 μM, 20 μM, 100 μM) for 40 min. Figure 11 (c) Single / two-photon combined image of the OTA probe after incubation with NTR and Cys. (Single-photon confocal imaging: Ex = 560nm, Em = 800-900nm; Two-photon confocal imaging: Ex = 780nm, Em = 450-550nm.)

[0069] Depend on Figure 11It was found that under single-photon excitation mode, HeLa cells cultured under oxygen-enriched (20% PO2) conditions showed almost no fluorescence after incubation with the probe OTA (10 μM). Conversely, HeLa cells cultured under hypoxic (5% PO2, 1% PO2) conditions showed bright intracellular fluorescence after incubation with OTA (10 μM), indicating that hypoxic cells, compared to oxygen-enriched cells, have a higher expression of nitroreductase. Under two-photon excitation mode, adding different concentrations of Cys to hypoxic (1% PO2) cells showed that the fluorescence intensity in HeLa cells increased with increasing Cys concentration. These cell experiments demonstrate that the probe OTA can not only respond continuously to NTR and Cys in a dual-mode manner, but also accurately detect cellular hypoxia through the combined monitoring of these two disease markers.

[0070] Example 7

[0071] The near-infrared two-photon dual-mode linked response NTR&Cys fluorescent probe (OTA) prepared in Example 1 was used for tissue detection, specifically as follows:

[0072] Two OTA excitation modes were used to perform sequential imaging of NTR and Cys in mouse liver tissue. The results are as follows: Figure 12 As shown, where Figure 12 (a) Single-photon excited near-infrared fluorescence images of the probe OTA (10 μM) in mouse liver tissue with different concentrations of NTR (0 μg / mL, 2 μg / mL, 5 μg / mL) in the presence of NADH (500 μM); Figure 12 (b) Two-photon excitation visible fluorescence images of mouse liver tissue after the probe OTA (10 μM) was incubated with 5 μg / mL NTR in the presence of NADH (500 μM) and then different concentrations of Cys (0 μM, 30 μM, 100 μM) were added. Figure 12 (c) Images of the probe OTA after incubation with NTR and Cys in single-photon near-infrared mode, two-photon visible light mode, and single-photon combined image; single-photon confocal imaging: Ex = 560nm, Em = 800-900nm, scale bar 60μm; two-photon confocal imaging: Ex = 780nm, Em = 450-550nm, scale bar 110μm.

[0073] Depend on Figure 12It was observed that in single-photon mode, almost no fluorescence was observed in tissues without NTR, while the fluorescence of both groups with NTR was significantly enhanced, and the intracellular fluorescence intensity increased with increasing NTR concentration. In two-photon mode, almost no fluorescence was observed in liver sections without Cys, while the fluorescence intensity was significantly enhanced after adding a certain concentration of Cys, and the fluorescence intensity gradually increased with increasing Cys concentration. These tissue imaging experiments demonstrate that the probe OTA can continuously trace NTR and Cys in liver tissue through dual-channel dual-excitation mode.

Claims

1. A near-infrared two-photon dual-mode linked response NTR and Cys fluorescent probe, characterized in that, Its structural formula is shown below: 。 2. A method for preparing the near-infrared two-photon dual-mode linked response NTR and Cys fluorescent probe as described in claim 1, characterized in that, Includes the following steps: (1) Anhydrous potassium carbonate was dissolved in water, deoxygenated and recrystallized, and transferred to a deoxygenated acetone solution of 4-(4-hydroxyphenyl)cyclohexanone and p-nitrobenzyl bromide. The solution was heated to reflux and cooled to room temperature after the reaction was completed. The solution was filtered and treated with activated carbon and concentrated at low temperature in the dark to obtain a yellow solid crude product. The crude product was recrystallized with petroleum ether and purified to obtain compound 2. The low temperature was -5~-10℃. (2) Freshly distilled DMF and CH2Cl2 were placed in a cold trap and stirred. Then, they were added to a mixture of CH2Cl2 and POCl3 and gradually raised to room temperature. Then, they were added dropwise to compound 2. The resulting mixture was heated under reflux and the reaction was monitored until complete. The reaction solution was placed in ice water overnight in the dark. It was extracted with a mixture of ethyl acetate-methanol-dichloromethane, dried with anhydrous Na2SO4 and vacuum evaporated to obtain compound 3. The temperature of the cold trap was -5 to -10℃. (3) Mix compound 3 with sodium acetate and add acetic anhydride and stir thoroughly to obtain a mixture. Add acetic anhydride solution of compound 1 dropwise to the mixture. Place the resulting mixture under light-protected conditions and increase the temperature gradually. After the reaction is completed, concentrate under reduced pressure and wash with a mixture of dichloromethane and methanol to obtain compound 4. (4) Under helium protection, 6-hydroxy-2-naphthaldehyde and NaH were dissolved in freshly distilled DMF and stirred. The DMF solution of compound 4 was slowly added and the reaction was carried out at room temperature in the dark. After the reaction was completed, the reaction solution was extracted with a mixture of dichloromethane and ethyl acetate in the dark. The extract was placed at low temperature and allowed to stand. The organic layer was dried with anhydrous Na2SO4 and the solvent was removed by vacuum pressure to obtain the crude product. After purification by silica gel column, the near-infrared two-photon two-mode linkage response NTR and Cys fluorescent probe were obtained. The reaction formula for preparing near-infrared two-photon dual-mode linked response NTR and Cys fluorescent probes is as follows: 。 3. The method for preparing near-infrared two-photon dual-mode linked response NTR and Cys fluorescent probes according to claim 2, characterized in that, In step (1), the molar ratio of 4-(4-hydroxyphenyl)cyclohexanone to 4-nitrobenzyl bromide is 3:4; in step (2), the ratio of POCl3 to compound 2 is 1 mL: 1 g; in step (3), the molar ratio of compound 3 to compound 1 is 1:2; in step (4), the molar ratio of 6-hydroxy-2-naphthaldehyde, NaH to compound 4 is 5:5:

1.

4. The method for preparing near-infrared two-photon dual-mode linked response NTR and Cys fluorescent probes according to claim 2, characterized in that, In step (2), the volume ratio of DMF, CH2Cl2 and POCl3 is 1:2:1; in step (3), the mass ratio of compound 3 to sodium acetate and acetic anhydride in the mixture is 1:1:1, and the mass ratio of compound 1 to acetic anhydride in the acetic anhydride solution of compound 1 is 3.2:

1.

5. The method for preparing near-infrared two-photon dual-mode linked response NTR and Cys fluorescent probes according to claim 2, characterized in that, In step (1), the reaction temperature of heating and reflux is 60℃ and the reaction time is 13 h; in step (3), the gradient heating condition is to heat up to 70℃ in 4 h; in step (4), the reaction time at room temperature in the dark is 6 h.

6. The method for preparing near-infrared two-photon dual-mode linked response NTR and Cys fluorescent probes according to claim 2, characterized in that, In step (2), the volume ratio of ethyl acetate, methanol, and dichloromethane in the ethyl acetate-methanol-dichloromethane mixture is 1:8:1; in step (3), the volume ratio of dichloromethane to methanol in the dichloromethane-methanol mixture is 10:1; in step (4), the volume ratio of dichloromethane to ethyl acetate in the dichloromethane-ethyl acetate mixture is 4:1.

Images