A lysosome-targeted near-infrared fluorescent probe for detecting hydroxyl radicals, and its preparation method and application

Abstract

A lysosomal-targeted near-infrared fluorescent probe for detecting hydroxyl radicals, and its preparation method and application. The structure of the fluorescent probe is shown in Formula I. The fluorescent probe provided by the present invention has excellent lysosomal targeting ability and can detect hydroxyl radicals with high sensitivity and selectivity in the near-infrared spectral wavelength region of 655nm. Furthermore, this near-infrared fluorescence response can enhance the tissue penetration ability of the fluorescent signal, reduce photodamage to biological samples, and avoid interference from biological background fluorescence signals, facilitating fluorescence imaging analysis of hydroxyl radicals in biological samples. Furthermore, the fluorescent probe provided by the present invention is simple to prepare, can be mass-produced, is stable, easy to store, has good biocompatibility, and is easy to use. It has great application prospects in the fields of chemistry, biology, and medicine. #imgabs0#

Description

Technical Field

[0001] The present invention relates to the technical field of biochemical analysis, and in particular to a lysosome-targeted near-infrared fluorescent probe for detecting hydroxyl radicals, and a preparation method and application thereof. Background Art

[0002] The hydroxyl radical (·OH) is one of the most destructive free radical species in organisms. It possesses extremely strong oxidative, dehydrogenative, and hydroxylation properties, causing irreversible damage to most biomolecules (including lipids, proteins, and nucleic acids), leading to cellular regulatory disturbances and closely implicated in the development and progression of major diseases such as cancer, cardiovascular disease, atherosclerosis, and neurodegenerative disorders. However, due to the high reactivity, short lifetime (nanoseconds), and low concentration (nanomolar) of ·OH, its accurate, efficient, real-time, and in situ analysis remains challenging, and its precise pathogenic role remains largely unresolved. The generation of ·OH in organisms primarily relies on the Fenton reaction, the Haber-Weiss reaction, and low-valent metal autooxidation reactions, all of which require the participation of free redox-active metals (such as iron and copper). Lysosomes are acidic organelles covered by a single membrane that contain dozens of hydrolases that can participate in the degradation of endogenous or exogenous biomolecules and the removal of damaged or aged cellular components. It can be seen that lysosomes play the role of a "distribution center" for intracellular material metabolism. For example, endogenous or exogenous iron-containing components of the cell (such as ferritin, damaged mitochondria, etc.) will be degraded in the lysosomes, causing a large amount of iron to accumulate in the lysosomes. The acidic microenvironment of the lysosomes themselves (pH ~4.5-5.0) may accelerate the Fenton reaction involving iron, leading to the production of ·OH. Therefore, lysosomes are an important site of ·OH production in cells. The development of highly sensitive and selective ·OH detection tools to achieve in situ non-destructive detection of ·OH in lysosomes has important research and practical significance.

[0003] Electron spin resonance spectroscopy, high-performance liquid chromatography, ultraviolet spectroscopy, electrochemical sensing and other technologies have been used for the detection of OH in vitro. However, the limitations of these methods are that they are not sensitive enough for the detection of OH, have low selectivity, require sophisticated instruments, are difficult to operate, and are not suitable for in situ imaging analysis of biological systems. In contrast, fluorescent probe imaging analysis technology has good application prospects due to its advantages such as high sensitivity, low cost, simple operation, non-invasive imaging, real-time imaging, and ultra-high temporal and spatial resolution, and is highly favored by researchers. Currently, OH fluorescent probes such as APF, HPF, CM-H2DCFDA, and 3-CCA are available on the market. However, these probes are often subject to oxidative interference from other reactive oxygen species (such as hydrogen peroxide, hypochlorous acid, and peroxynitrite), or have low analytical sensitivity and short response wavelength, which are not conducive to imaging analysis of biological samples. Moreover, these probes cannot achieve lysosomal targeted analysis. Summary of the Invention

[0004] The present invention aims to provide a lysosome-targeted near-infrared fluorescent probe for detecting ·OH, and a preparation method and application thereof.

[0005] To this end, in a first aspect, the present invention provides a lysosomal-targeted near-infrared fluorescent probe for detecting ·OH, the structure of which is shown in Formula I:

[0006]

[0007] In a second aspect, the present invention provides a method for preparing a fluorescent probe of Formula I, comprising the following reaction steps: (1) a nucleophilic substitution reaction is performed on compound 1 and compound 2 in an organic solvent to prepare an intermediate compound 3; (2) a condensation reaction is performed on compound 3 and compound 4 in an organic solvent under base catalysis to prepare an intermediate compound 5; (3) an electrophilic addition reaction is performed on compound 5 and compound 6 in a water / organic mixed solvent under base catalysis to obtain the fluorescent probe of Formula I; see the following reaction scheme,

[0008]

[0009] Furthermore, the organic solvent in step (1) is at least one of acetonitrile, N,N-dimethylformamide and dimethyl sulfoxide, the reaction temperature is 80-130° C., and the reaction time is 1-24 h; the organic solvent in step (2) is at least one of methanol, ethanol and acetic acid, the base is at least one of sodium acetate, potassium carbonate, pyridine and piperidine, the reaction temperature is 30-120° C., and the reaction time is 1-24 h; the organic solvent in step (3) is at least one of acetonitrile, ethanol or methanol, the base is at least one of sodium hydroxide, potassium hydroxide, sodium carbonate or potassium carbonate, the reaction temperature is 30-100° C., and the reaction time is 1-48 h.

[0010] In a third aspect, the present invention provides an application of the fluorescent probe in ·OH detection.

[0011] Specifically, ·OH is detected by the change in spectral signal caused by the expansion of the conjugated system after the probe reacts with ·OH, as shown in the following response mechanism:

[0012]

[0013] Specifically, the fluorescence response intensity of the fluorescent probe at a wavelength of 655 nm is detected to determine OH. The fluorescent probe provided by the present invention has the following characteristics and advantages:

[0014] 1) After reacting with OH, the fluorescence emission wavelength red-shifts to the near-infrared wavelength region of 655 nm, which can enhance the tissue penetration ability of the light signal, reduce photodamage to biological samples, and avoid interference from biological background fluorescence signals;

[0015] 2) The probe can selectively target the cell lysosome, enabling in situ non-destructive fluorescence imaging analysis of ·OH in the lysosome;

[0016] 3) The probe has good selectivity for ·OH analysis and is not interfered with by other reactive oxygen species or physiologically active species;

[0017] 4) The probe has high analytical sensitivity to ·OH and can respond to trace levels of ·OH.

[0018] 5) Good biocompatibility and easy to use;

[0019] 6) The preparation method is simple, can be produced in large quantities, and is easy to store. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Various other advantages and benefits will become apparent to those skilled in the art by reading the detailed description of the preferred embodiment below. The accompanying drawings are only for the purpose of illustrating the preferred embodiment and are not to be considered as limiting the present invention. In the accompanying drawings:

[0021] Figure 1 Graph showing the fluorescence response of the fluorescent probe shown in Formula I to different concentrations of TCBQ / H2O2 reagents (generating ·OH).

[0022] Figure 2 This is a linear relationship diagram of the fluorescence response intensity of the fluorescent probe shown in Formula I to the TCBQ / H2O2 reagent at a wavelength of 655 nm and the concentration of the TCBQ / H2O2 reagent.

[0023] Figure 3Graph showing the fluorescence response intensity of the fluorescent probe represented by Formula I to different potential interfering substances (including common reactive oxygen species and physiological substances) at a wavelength of 655 nm.

[0024] Figure 4 The fluorescent probe shown in formula I is sensitive to different concentrations of low-valent metal ions (Fe 2+ or Cu + ) Fluorescence response of trace ·OH produced during autooxidation.

[0025] Figure 5 This is the fluorescence imaging diagram of the fluorescent probe shown in Formula I on ·OH in HeLa cells under PMA stimulation conditions.

[0026] Figure 6 This is a co-localization imaging diagram of the fluorescent probe shown in Formula I and the commercial lysosomal dye Lyso-tracker Green. DETAILED DESCRIPTION

[0027] Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments described herein. Rather, these embodiments are provided to enable a more thorough understanding of the present disclosure and to fully convey the scope of the present disclosure to those skilled in the art.

[0028] Example 1 Preparation of the fluorescent probe represented by formula I

[0029] This embodiment provides a method for preparing the fluorescent probe represented by Formula I, and its synthetic route is as follows:

[0030]

[0031] The specific steps are as follows:

[0032] Compound 1 (1.4 g, 10 mmol) and compound 2 (0.96 g, 11 mmol) were placed in a round-bottom flask, and 10 mL of dimethyl sulfoxide (DMSO) was added to dissolve the solid. The flask was placed in an oil bath at 100°C for 16 hours, and 25 mL of deionized water was added to the reaction mixture to quench the reaction. Ethyl acetate (EA) (10 mL × 3) was then added for extraction. The organic phase was washed with a saturated aqueous sodium chloride solution and dried over anhydrous magnesium sulfate. The solid residue obtained after removing the organic phase by distillation under reduced pressure was purified by silica gel column chromatography (silica gel G, 200-300 mesh) using PE / EA (1:1, v / v) as eluent to obtain the intermediate compound 3 (1.6 g, 7.7 mmol, 82% yield, yellow solid). 1H NMR (400MHz, 298K, DMSO-d6): δ11.10 (s, 1H), 9.76 (s, 1H), 7.50 (d, J = 8.9Hz, 1H), 6.60 (d,J=9.0Hz,1H),6.32(d,J=2.3Hz,1H),3.70(t,J=4.9Hz,4H),3.32(t,J=5.0Hz,4H). 13 C NMR (101 MHz, DMSO-d6): δ191.64, 163.43, 157.00, 133.56, 113.60, 106.56, 99.22, 66.19, 46.80. Compound 3 (1.04 g, 10 mmol) and compound 4 (1.4 g, 13 mmol) were placed in a round-bottom flask, and 200 mL of anhydrous ethanol and 2 mL of piperidine were added successively. The round-bottom flask was placed in an 80°C oil bath and refluxed for 4 hours. The reaction was then transferred to an ice-water bath, and an orange-yellow needle-like precipitate gradually formed. After filtration and drying, the intermediate compound 5 (2.5 g, 9.2 mmol, yield 67%) was obtained. 1 H NMR (400MHz, Chloroform-d): δ8.45 (s, 1H), 7.47 (d, J = 8.9Hz, 1H), 6.83 (d, J = 2.4Hz, 1H), 6.67 (d, J = 2.4Hz, 1H), 3.87 (t, J = 5.0Hz, 4H), 3.39 (t, J = 5.0Hz, 4H), 2.69 (s, 3H). 13 C NMR (101MHz, Chloroform-d): δ195.70,160.36,158.12,155.41,147.72,131.55,118.62,111.40,110.05,99.31,66.29,46.99,30.66.

[0033] Compound 5 (0.74 g, 2 mmol), compound 6 (0.6 g, 2 mmol) and sodium hydroxide (130 mg, 3.25 mmol) were placed in a round-bottom flask, and 20 mL of ethanol and 20 mL of water were added. The mixture was reacted in a water bath at 32°C under dark conditions for 24 hours. The solvent was removed by distillation under reduced pressure to obtain a crude product, which was then purified by silica gel column chromatography (silica gel G, 200-300 mesh) using DCM / MeOH (100:1, v / v) as eluent to obtain the fluorescent probe represented by Formula I (0.13 g, 0.3 mmol, 15%, orange powder). 1H NMR (400MHz, DMSO-d6): δ10.19(s,1H),9.05(s,1H),8.62(s,1H),8.40(s,1H ),7.68(d,J=8.9Hz,1H),7.50(s,1H),7.32(d,J=7.6Hz,1H),7.18(t,J=7.5H z,1H),6.99(d,J=8.0Hz,1H),6.80(s,1H),4.50(t,J=5.9Hz,1H),3.97(s,3H ), 3.73 (t, J = 4.8Hz, 4H), 3.42 (t, J = 5.1Hz, 4H), 2.90 (dd, J = 15.6, 5.2Hz, 1H). 13 C NMR (101MHz, DMSO-d6): δ195.20,188.71,187.17,176.17,159.76,157.92,155.66,147.87,140.71,138.58,128.9 0,127.05,126.16,124.34,116.44,113.85,111.76,109.59,98.66,66.13,55.37,51.31,46.90,30.57.HR-MS:m / z calcd.for[C26H24N2O5+Na] + ,467.1577;found,467.1586.

[0034] Example 2 Determination of Hydroxyl Radicals by the Fluorescent Probe Represented by Formula I in Phosphate Buffer

[0035] Add 3 mL of 20 mM phosphate buffer to a 5 mL test tube, add 30 μL of probe solution to make the final probe concentration 10 μM, and finally add a series of TCBQ / H2O2 reagents (which can generate hydroxyl radicals) with different concentrations. After reacting for 30 minutes, measure the fluorescence spectrum with an excitation light wavelength of 510 nm.

[0036] Figure 1 The fluorescence spectrum response diagram of the fluorescent probe shown in formula I to different concentrations of TCBQ / H2O2 reagent is shown in FIG. Figure 1 It can be seen that with the increase of TCBQ / H2O2 concentration, the fluorescence of the probe at 655nm wavelength gradually increases; Figure 2 The relationship between fluorescence intensity and TCBQ / H2O2 reagent concentration was obtained by plotting the fluorescence intensity at 655 nm as the ordinate and the TCBQ / H2O2 concentration as the abscissa. Figure 2 It can be seen that in the range of 0-20 μM, the fluorescence intensity shows a good linear relationship with the concentration of TCBQ / H2O2 reagent.

[0037] Example 3 Analytical selectivity of the fluorescent probe represented by formula I for ·OH

[0038] A phosphate buffer solution containing 10 μM probe was added to a 5 mL test tube, and then 100 μM of different common reactive oxygen species (including NO, OCl - 、H2O2、 1 O2, O2 ·- 、ONOO - and TBHP), 1 mM metal ions, 1 mM amino acids, 5 mM glutathione, 10 mM glucose, or 100 μg / mL BSA. The fluorescence spectrum was measured after 30 min of reaction with an excitation wavelength of 510 nm.

[0039] The results are as attached Figure 3 As shown, other reactive oxygen species, except ·OH, do not induce a significant fluorescence response from the fluorescent probe represented by Formula I, even at high concentrations (five times that of ·OH). Common bioactive species, such as various metal ions, GSH, amino acids, glucose, and proteins, also do not induce a fluorescence response from the fluorescent probe represented by Formula I. These results demonstrate that the fluorescent probe represented by Formula I has good analytical selectivity for ·OH.

[0040] Example 4: Detection of trace ·OH produced during the self-oxidation of low-valent metals by the fluorescent probe represented by formula I

[0041] Phosphate buffer containing 10 μM probe was added to a 5 mL test tube, and then different concentrations of Fe 2+ or Cu + (0–100 μM), and the fluorescence spectrum was measured after 30 min of reaction. The excitation wavelength was 510 nm.

[0042] The self-oxidation process of low-valent metals refers to the process of low-valent metal ions (such as Fe 2+ and Cu + ) directly oxidizes with oxygen molecules. This process can generate trace amounts of ·OH through a series of reaction steps, and the generated ·OH cannot be directly detected by commonly used free radical detection methods (such as electron spin resonance spectroscopy). Therefore, this embodiment provides the application of the fluorescent probe represented by Formula I to detect trace amounts of ·OH generated during the self-oxidation of low-valent metals. The results are shown in the attached figure. Figure 4 As shown, with different concentrations of Fe 2+ or Cu + After 30 min of reaction (0–100 μM), the fluorescence of the probe at a wavelength of 655 nm gradually increased, indicating that the fluorescent probe represented by Formula I can be used to detect trace amounts of ·OH produced during the self-oxidation of low-valent metals with good analytical sensitivity.

[0043] Example 5 Detection of Endogenous ·OH Level Changes in HeLa Cells Induced by Phorbol 12-Myristate 13-Acetate (PMA) Using the Fluorescent Probe Represented by Formula I

[0044] 1) HeLa cells were cultured in DMEM medium containing 10% (v / v) fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin in an environment of 37°C, 5% (v / v) CO2, and 96% humidity. The cells were cultured in a glass-bottomed dish designed for laser confocal fluorescence microscopy for 24 h to allow the cells to fully adhere to the dish bottom.

[0045] 2) Add 2 μL of a 1 mg / mL DMSO solution of PMA to the cell culture dish to a final PMA concentration of 2 μg / mL. Incubate the cells for 2, 4, or 6 h to induce endogenous ·OH production. To scavenge the generated ·OH, add 200 μM Tempol for 30 min after 6 h of PMA stimulation.

[0046] 3) Aspirate the old culture medium, wash the cells with PBS buffer, then add 1 mL of DMEM culture medium containing 10 μM probe. Incubate for 30 min before performing fluorescence imaging on a laser confocal fluorescence microscope. The ·OH channel is excited by a 561 nm laser, and the fluorescence signal is collected in the range of 600-700 nm. Changes in signal intensity represent changes in ·OH levels.

[0047] PMA is a commonly used inducer of intracellular reactive oxygen species, which can cause cells to produce a large amount of reactive oxygen species (including ·OH). Figure 5 The fluorescence imaging diagram of the fluorescent probe shown in formula I on HeLa cells treated with PMA is shown in FIG. Figure 5 It can be seen that with the extension of PMA incubation time, the fluorescence signal of the ·OH channel gradually increased, indicating that the PMA induction process is accompanied by an increase in the ·OH level. It also shows that the fluorescent probe represented by Formula I can be used for in situ non-destructive fluorescence imaging monitoring of intracellular ·OH.

[0048] Example 6 Co-localization Imaging of the Fluorescent Probe Represented by Formula I and Commercial Lysosomal Dyes

[0049] After HeLa cells were cultured in glass-bottomed culture dishes for 24 h, the old culture medium was aspirated and the cells were washed with PBS buffer. Then, 1 mL of DMEM culture medium containing 10 μM probe and 100 nM commercial lysosomal dye Lyso-tracker Green was added and incubated for 30 min. Then, 100 μM Fenton reagent (exogenous ·OH) was added and incubated for 30 min. Finally, fluorescence imaging was performed.

[0050] As attached Figure 6As shown, in HeLa cells, the red channel fluorescence of the fluorescent probe shown in I (a) and the green channel fluorescence of Lyso-trackerGreen (b) have a good overlap effect (c), and the Pearson coefficient of the intensity correlation diagram of the two channels is 0.88 (e), indicating that the fluorescent probe shown in I has good lysosomal targeting ability.

[0051] Finally, it should be noted that the above examples only illustrate the preparation method and some applications of one fluorescent probe represented by Formula I, and the remaining preparation methods, analysis and testing, and imaging application conditions are not listed one by one.

[0052] The foregoing description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications or substitutions that can be readily conceived by a person skilled in the art within the technical scope disclosed herein are intended to be encompassed within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be subject to the scope of protection of the claims.

Claims

1. A lysosome-targeted near-infrared fluorescent probe for detecting hydroxyl radicals, characterized in that: The structure of the fluorescent probe is shown in Formula I, 。 2. The method for preparing a fluorescent probe according to claim 1, wherein: The method comprises the following reaction steps: (1) a nucleophilic substitution reaction is carried out between compound 1 and compound 2 in an organic solvent to prepare an intermediate compound 3; (2) a condensation reaction is carried out between compound 3 and compound 4 in an organic solvent under base catalysis to prepare an intermediate compound 5; (3) an electrophilic addition reaction is carried out between compound 5 and compound 6 in a water / organic mixed solvent under base catalysis to obtain a fluorescent probe shown in formula I; See the following reaction scheme, 。 3. The preparation method according to claim 2, wherein The organic solvent in step (1) is at least one of acetonitrile, N,N-dimethylformamide and dimethyl sulfoxide, the reaction temperature is 80-130 °C, and the reaction time is 1-24 h; the organic solvent in step (2) is at least one of methanol, ethanol and acetic acid, the base is at least one of sodium acetate, potassium carbonate, pyridine and piperidine, the reaction temperature is 30-120 °C, and the reaction time is 1-24 h; the organic solvent in step (3) is at least one of acetonitrile, ethanol or methanol, the base is at least one of sodium hydroxide, potassium hydroxide, sodium carbonate or potassium carbonate, the reaction temperature is 30-100 °C, and the reaction time is 1-48 h.

4. Use of the fluorescent probe as claimed in claim 1 in preparing a hydroxyl radical detection probe.

5. The use according to claim 4, characterized in that: The hydroxyl radical detection probe detects hydroxyl radicals by monitoring the fluorescence signal intensity at 655 nm.

6. The use according to claim 4, characterized in that: Hydroxyl radical detection probes are used for environmental sample detection.

7. The use according to claim 4, characterized in that: Hydroxyl radical detection probes are used for bioimaging detection.

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