A method for detecting endogenous H2S n Activatable dual-color fluorescent probe and its preparation method and application

Abstract

The present invention belongs to the field of biochemistry and specifically discloses a method for detecting endogenous H2S n An activatable dual-color fluorescent probe for the highly chemically selective detection of H2S n The nanoprobe structure is a small molecule probe BOD-CN encapsulated in the hydrophobic cavity of micelles formed by mPEG-DSPE2000. BOD-CN is obtained by finely regulating the electrophilicity of BODIPY luminophores based on a strategy without masking groups, and it can specifically detect H2S. n And there are no toxic by-products. The encapsulated nanoprobes are not only n It has high chemical selectivity and exhibits a rare aggregation-induced dual-color fluorescence response. By using the information complementation of this activatable dual-color fluorescence channel, it successfully achieved intracellular H2S n High-integrity imaging.

Description

Technical Field

[0001] The present invention belongs to the field of biochemistry and relates to a fluorescent probe and its synthesis and application, which can realize the selective detection of H2S n , specifically, it relates to the synthesis methods and cell imaging applications of nanoprobes. Background Art

[0002] Reactive sulfur species are a class of sulfur-containing molecules that play an important role in biological systems. Among them, hydrogen sulfide (H2S), as a widely studied gaseous signaling molecule among reactive sulfur species, is believed to play an important regulatory role in many pathological and physiological processes. In contrast, polysulfides (H2S), derivatives produced by the interaction between endogenous H2S and reactive oxygen species / enzymes, are n ; n>1) have attracted much less attention. In fact, new evidence suggests that some of the biological effects previously reported to be associated with H2S are actually caused by H2S n Produced or mediated by H2S n It has stronger nucleophilicity and reducing properties than other biothiols and H2S. n It has a great effect, but it has a great effect on H2S. n The study of the exact mechanism of action in biology is still in its early stages, and many questions remain to be answered.

[0003] Although there are many H2S n Activatable fluorescent probes have been developed and have gradually overcome some of the limitations of early probes, but these design strategies mainly rely on several H2S n activatable protecting groups and require careful design of linker groups. Therefore, many fluorophores are used in H2S based on this masking strategy. n The design of activatable probes is limited. At the same time, such masking group-based probes may produce potential non-fluorescent and toxic byproducts when activated, which is not conducive to the detection of H2S n In addition, although some functional materials have been used to successfully realize multi-color fluorescence imaging and greatly improve the imaging quality, it is still a huge challenge to design activatable multi-color emission molecular probes. n Activatable multicolor fluorescent organic molecular probes will provide a new tool for exploring H2S n Provide high-performance research tools for mediated biological events.

[0004] Based on this background, the present invention designed and synthesized a nanoprobe with special activatable aggregation-induced dual-color fluorescence, which showed a strong affinity for H2S. nThe dual-color fluorescence response of the molecule, which turns on bright fluorescence emission at 588 nm and 750 nm, enables high-quality imaging of H2S in living cells based on two different fluorescence channels. n . Summary of the Invention

[0005] The first object of the present invention is to provide a method for detecting endogenous H2S n activatable dual-color fluorescent probe.

[0006] The second object of the present invention is to provide a method for detecting endogenous H2S n Preparation method of activatable dual-color fluorescent probe.

[0007] The third object of the present invention is to provide a method for selectively detecting H2S n The nanoprobe is used to detect endogenous hydrogen polysulfide in cells using dual-color fluorescence response.

[0008] The present invention is achieved through the following technical solutions:

[0009] The present invention provides a method for detecting endogenous H2S n The activatable dual-color fluorescent probe has a nanoprobe structure in which the small molecule probe BOD-CN is encapsulated in the hydrophobic cavity of micelles formed by mPEG-DSPE2000.

[0010] The structure of BOD-CN is shown in Formula I:

[0011]

[0012] The present invention provides the above nanoprobe with strong near-infrared fluorescence emission at 588nm and 750nm, and uses it to detect endogenous H2S in Hela cells. n Fluorescence imaging.

[0013] The invention has the function of selectively detecting H2S n The nanoprobe has a dye molecule BOD-CN and a PEG hydrophobic cavity inside and a PEG hydrophilic long chain outside.

[0014] The nanoprobe of the present invention utilizes dual-color fluorescence response to achieve information complementarity in different channels, thereby providing an image with higher integrity.

[0015] The present invention also provides the method for selectively detecting H2S n The preparation method of the nanoprobe is as follows:

[0016]

[0017] The specific method is:

[0018] 1. Synthesis of Compound A

[0019] Synthesis of BOD-CHO Reference Patent - Zhao Chunchang, Zhang Xiuli, Zhang Lili, Wang Feiyi, Jiang Haijun, An Jiancai, Zhang Fan, A fluorescent probe for detecting endogenous H2S and its preparation method and application, China: Patent No.: ZL201410766204.6, 2015-03-11, Certificate No.: No. 2250092.

[0020] 2. Synthesis of fluorescent probe BOD-CN

[0021] Malononitrile was dissolved in anhydrous ethanol, and then the compound BOD-CHO was added. The mixture was stirred, heated, and refluxed under argon for 12 hours. After cooling to room temperature, the product BOD-CN was obtained after post-treatment as a red solid.

[0022] 3. Synthesis of Nanoprobe BOD-CN-NP

[0023] BOD-CN was quickly added to 2 mL of deionized water in which mPEG-DSPE was dissolved, sonicated for 10 min, and then dialyzed to obtain a nanoprobe solution.

[0024] The present invention also provides a method for selectively detecting H2S n Nanoprobes for detecting endogenous H2S n Application on.

[0025] Beneficial technical effects of the present invention:

[0026] The nanoprobe provided by the present invention has a simple synthesis method and relatively low cost, but has excellent imaging performance and can image H2S in living cells with high integrity based on the dual-color fluorescence response of two different fluorescence channels. n . BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the synthetic route of the small molecule organic probe BOD-CN.

[0028] Figure 2 This is the construction process of the nanoprobe BOD-CN-NP.

[0029] Figure 3 The small molecule organic probe BOD-CN in CDCl3 1 HNMR spectrum.

[0030] Figure 4 Structural characterization of the nanoprobe BOD-CN-NP by (a) transmission electron microscopy and (b) dynamic light scattering.

[0031] Figure 5The small molecule organic probe BOD-CN has a high sensitivity to H2S at a concentration of 10 μmol / L. n UV absorption spectrum changing with time. (Note: H2S n The concentration was 100 μmol / L, the test solvent was PBS / MeCN, v / v, 1:1, pH = 7.4).

[0032] Figure 6 The nanoprobe BOD-CN-NP has a concentration of 10 μmol / L and a high sensitivity to H2S n UV absorption spectrum changing with time. (Note: H2S n The concentration was 100 μmol / L, and the test solvent was PBS, pH = 7.4).

[0033] Figure 7 The nanoprobe concentration is 10 μmol / L for H2S n Fluorescence emission spectrum changing with time. (Note: H2S n The concentration was 100 μmol / L, the test solvent was PBS, pH = 7.4, the excitation wavelength was 490 nm (Figure a), 550 nm (Figure b), and 670 nm (Figure c).

[0034] Figure 8 Nanoprobes for imaging endogenous H2S in HeLa cells using activated dual-color fluorescence n 's effect diagram. DETAILED DESCRIPTION

[0035] The present invention will be further described below with reference to the following examples. Those skilled in the art should understand that the examples are only for illustration and do not constitute any limitation to the present invention.

[0036] Example 1

[0037] Synthesis of small molecule organic probe represented by formula Ⅰ

[0038]

[0039] Synthesis of compound A

[0040] Synthesis reference patent for compound A - Zhao Chunchang, Zhang Xiuli, Zhang Lili, Wang Feiyi, Jiang Haijun, An Jiancai, Zhang Fan, A fluorescent probe for detecting endogenous H2S and its preparation method and application, China: Patent No.: ZL201410766204.6, 2015-03-11, Certificate No.: No. 2250092.

[0041] Synthesis of fluorescent probe BOD-CN

[0042] Dissolve 20 mg (0.3 mmol) of malononitrile in 20 mL of anhydrous ethanol, then add 39 mg (0.1 mmol) of the compound BOD-CHO. The mixture was stirred, heated, and refluxed under argon for 12 hours. Cool to room temperature, dilute with 30 mL of DCM, and combine the organic phases, wash three times with deionized water, dry over anhydrous sodium sulfate, and remove the solvent by rotary evaporation under reduced pressure. The crude product was purified by silica gel column chromatography to yield a red solid (26 mg, 61%). 1 HNMR (400MHz, CDCl3) δ = 7.68 (s, 1H), 7.57-7.52 (m, 3H), 7.33-7.31 (dd, 2H), 7.04(s,1H),2.70(s,3H),2.43-2.38(q,2H),1.51(s,3H),1.08-1.04(t,3H).

[0043] Figure 5 The small molecule organic probe BOD-CN has a high sensitivity to H2S at a concentration of 10 μmol / L. n UV absorption spectrum changing with time. (Note: H2S n The concentration was 100 μmol / L, the test solvent was PBS / MeCN, v / v, 1:1, pH = 7.4).

[0044] Example 2

[0045] Synthesis of nanoprobe BOD-CN-NP

[0046] BOD-CN (0.2 mg) was quickly added to 2 mL of deionized water in which mPEG-DSPE (20 mg) was dissolved, and the mixture was sonicated for 10 minutes and then dialyzed for 6 hours to obtain a nanoprobe solution. Figure 4 .

[0047] Figure 4 Structural characterization of the nanoprobe BOD-CN-NP by (a) dynamic light scattering and (b) transmission electron microscopy, demonstrating the successful construction of the nanoprobe.

[0048] Example 3

[0049] Hela cells were seeded in cell culture dishes, allowed to adhere for 24 hours before the experiment, and washed with PBS buffer (pH 7.4) before imaging. The cells were incubated with 10 μM probe for the corresponding time and washed three times with PBS buffer before confocal imaging. When imaging endogenous agonists or scavengers, the cells were first incubated with LPS or ZnCl2 for the corresponding time, washed three times with PBS buffer, and then incubated with the probe. The nanoprobe BOD-CN-NP was directly diluted to the desired concentration in cell culture medium for cell imaging. Confocal imaging was performed using a Leica TCS SP8 and a 63× water objective. The red channel excitation wavelength was 633 nm, and the yellow channel excitation wavelength was 550 nm.

[0050] Figure 8 Nanoprobes for imaging endogenous H2S in HeLa cells using activated dual-color fluorescence n The effect diagram shows that BOD-CN-NP can be applied to H2S in living cells. n Imaging.

[0051] Figure 6 The nanoprobe BOD-CN-NP has a concentration of 10 μmol / L and a high sensitivity to H2S n UV absorption spectrum changing with time. (Note: H2S n The concentration was 100 μmol / L, the test solvent was PBS, pH = 7.4). UV absorption spectroscopy demonstrated that mPEG-DSPE micelles promoted the close molecular stacking of BOD-CN, and BOD-CN-NPs and H2S n After the response, two new absorption bands appeared, located at 550nm and 670nm. In contrast, without the assistance of mPEG-DSPE, BOD-CN only showed a dull and weak absorption at 550nm after activation ( Figure 5 ).

[0052] Figure 7 The nanoprobe concentration is 10 μmol / L for H2S n Fluorescence emission spectrum changing with time. (Note: H2S n The concentration was 100 μmol / L, the test solvent was PBS, pH = 7.4, and the excitation wavelength was 490 nm (Figure a), 550 nm (Figure b), and 670 nm (Figure c). This demonstrates that BOC-CN-NP can simultaneously detect H2S using two different fluorescence channels. n .

Claims

1. A method for detecting endogenous H2S n The activatable dual-color fluorescent nanoprobe is characterized by: Nanoprobe structure: The small molecule probe BOD-CN is encapsulated in the hydrophobic cavity of micelles formed by mPEG-DSPE2000; The BOD-CN structure is shown in Formula I: 。 2. A method for detecting endogenous H2S according to claim 1 n The activatable dual-color fluorescent nanoprobe is characterized by: The nanoprobe has a dye molecule BOD-CN and a PEG hydrophobic cavity inside and a PEG hydrophilic long chain outside.

3. A method for detecting endogenous H2S according to claim 1 n The activatable dual-color fluorescent nanoprobe is characterized by: The nanoprobe utilizes dual-color fluorescence response to achieve information complementarity in different channels, thereby providing an image with higher integrity.

4. A method for detecting endogenous H2S according to any one of claims 1 to 3 n The preparation method of the activatable dual-color fluorescent nanoprobe is as follows:

5. A method for detecting endogenous H2S according to any one of claims 1 to 3 n Activatable dual-color fluorescent nanoprobe for detecting endogenous H2S n For non-diagnostic applications.

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