阅读说明：本技术 一种检测硝基还原酶的双光子荧光探针及其制备方法和用途 (Two-photon fluorescent probe for detecting nitroreductase and preparation method and application thereof ) 是由 冯燕 汪旭东 陈德宝 周惠敏 卫丽云 于 2021-08-25 设计创作，主要内容包括：本发明公开了一种检测硝基还原酶的双光子荧光探针及其制备方法和用途,其中检测硝基还原酶的荧光探针的结构如下：本发明检测硝基还原酶的双光子荧光探针,在体外实验中表现出对硝基还原酶的良好响应性。细胞毒性测试表明该荧光探针的生物毒性较低,共聚焦荧光显微成像实验表明该荧光探针可以检测出常氧/缺氧时HeLa细胞线粒体内的硝基还原酶含量变化,可用于判断细胞线粒体内是否缺氧。(The invention discloses a two-photon fluorescent probe for detecting nitroreductase and a preparation method and application thereof, wherein the structure of the fluorescent probe for detecting nitroreductase is as follows: the two-photon fluorescent probe for detecting nitroreductase shows good responsiveness to nitroreductase in vitro experiments. The cytotoxicity test shows that the biological toxicity of the fluorescent probe is low, and the confocal fluorescence microscopic imaging experiment shows that the fluorescent probe can detect the content change of nitroreductase in HeLa cell mitochondria during normal oxygen/oxygen deficiency and can be used for judging whether the cell mitochondria are oxygen deficient.)

1. A two-photon fluorescent probe for detecting nitroreductase is characterized in that the structural formula is as follows:

2. a method for preparing the two-photon fluorescent probe according to claim 1, which comprises the steps of:

step 1: mixing Compound1 and p-hydroxybenzaldehyde, adding ethanol and piperidine under anaerobic condition, heating at 60 deg.C for 12h, reacting completely, and vacuum filtering with diethyl ether to obtain OH-1;

step 2: at 45 deg.C, adding OH-1 and Cs₂CO₃Mixing with 4-nitrobenzyl bromobenzene, dripping 10ml of DMF under the anaerobic condition, reacting for 4h at 45 ℃, purifying by column chromatography after complete reaction to obtain the target product NO₂-1；

The reaction scheme is as follows:

3. the method of claim 2, wherein:

in the step 2, the eluent used in the column chromatography purification is dichloromethane and petroleum ether, namely 2:1, v/v.

4. Use of the two-photon fluorescent probe according to claim 1, wherein:

the two-photon fluorescent probe is used for preparing a detection reagent for monitoring nitroreductase in living cell mitochondria.

5. Use according to claim 4, characterized in that:

the detection reagent has the capability of responding to the change of the nitroreductase in mitochondria in the process of cell hypoxia.

Technical Field

The invention relates to a two-photon fluorescent probe for detecting nitroreductase and a preparation method and application thereof, which are used for realizing the detection of the change of nitroreductase in cell mitochondria under oxygen deficiency and normal oxygen by two-photon confocal imaging and have the advantages of high selectivity, high sensitivity and low biological toxicity.

Background

Hypoxia is a condition of insufficient oxygen supply to tissues, and a large number of experiments show that hypoxia can be caused by tumors, wherein the tumors reduce oxygen transportation by reducing blood supply, and the tumors have larger oxygen consumption so as to cause hypoxia. Clinical research shows that the hypoxia condition of the tumor is closely related to the growth and the progress of the tumor, and the hypoxia makes the tumor more easily metastasize and deteriorate, so that the development of a new method for detecting the cell hypoxia is very significant for clinical research. Hypoxia causes many reduction reactions in the organism, resulting in the accumulation of a number of reducing enzymes including aldehyde oxidase, acyl dehydrogenase, and Nitroreductase (NTR). Nitroreductase has been extensively studied in recent years, and is an ideal hypoxia marker with high selectivity and representativeness. In the absence of oxygen, the nitroreductase can reduce a nitro-group-containing compound with Nicotinamide Adenine Dinucleotide (NADH) as an electron donor in the presence of NADH, and the nitroreductase content is abnormally increased, so that the nitroreductase has a close relationship with the level of oxygen deficiency in the organism.

The fluorescent probe has the advantages of rapid reaction, high sensitivity, high specificity and excellent biocompatibility, and is widely applied to the detection of nitroreductase. In recent years, many probes have made significant progress in detecting nitroreductase enzymes. However, such probes have little or no ability to localize mitochondria and have no two-photon properties for imaging cells. Two-photon confocal imaging has become an effective method for visually monitoring analytes at the sub-cellular level due to the advantages of providing deeper penetration and high resolution imaging. Therefore, the design of a two-photon fluorescent probe is very important for realizing the sensitive detection of nitroreductase in mitochondria of living cells.

Disclosure of Invention

The invention aims to provide a two-photon fluorescent probe for detecting nitroreductase as well as a preparation method and application thereof, and aims to solve the technical problem that an organic small molecular structure capable of specifically identifying nitroreductase is obtained through molecular design so as to monitor the change of the content of nitroreductase in mitochondria of living cells through two-photon confocal fluorescence imaging.

The two-photon fluorescent probe of the present invention is abbreviated as NO₂-1, having the formula:

the preparation method of the two-photon fluorescent probe comprises the following steps:

step 1: compound1(0.5g, 1.17mmol) and p-hydroxybenzaldehyde (1.25g, 10.23mmol) were placed in a Schlenk bottle, ethanol (10mL) and piperidine (2 drops) were added under oxygen-free conditions, heated at 60 ℃ for 12h, after completion of the reaction, cooled to room temperature, filtered with petroleum ether and vacuum dried to give a crimson solid OH-1, 0.4g, 59.25% yield.

Step 2: at 45 deg.C, OH-1(1g, 2.53mmol), Cs₂CO₃(1.5g, 3.06mmol) and 4-nitrobenzylborobenzene (0.82g, 3.8mmol) were placed in a Schlenk flask under N₂Adding 10ml DMF dropwise under the atmosphere, stirring at 45 deg.C for 4h, cooling to evaporate solvent, vacuum filtering with petroleum ether, purifying the obtained solid with chromatographic column (dichloromethane: petroleum ether ═ 2:1), distilling under reduced pressure, and vacuum drying to obtain light yellow powder NO₂-1, 0.6g, yield 64%.

The synthetic route of the two-photon fluorescent probe is as follows:

the two-photon fluorescent probe is used for preparing a detection reagent for detecting the change of nitroreductase in living cell mitochondria. The detection method comprises the following steps:

NO of the present invention₂-1 dissolving in DMSO (5mL) to obtain 2mM stock solution, taking 15. mu.L NO₂-1 mother liquor was added to 3mL of test solutions of nitroreductase at various concentrations. NO₂The single photon excitation wavelength of-1 is 500nm, the fluorescence spectrum change in the range of 520-640nm can be detected, the content of nitroreductase from 0 mu g/mL to 7 mu g/mL can be obviously observed, the fluorescence intensity is enhanced by 80 times at about 558nm, and the concentration of nitroreductase and the fluorescence intensity present good linear relation (R is²＝0.98)。

To test NO₂-1 reaction time for nitroreductase, 15. mu.L NO₂And (3) adding the mother liquor of the-1 into 3mL of nitroreductase solutions with different concentrations to obtain a final test solution. The excitation wavelength is the same as above, and the change of fluorescence spectrum in the range of 520-640nm is detected, so that NO can be observed when the time is 30min₂The fluorescence intensity of-1 substantially stabilized. The specific response capability of the probe to nitroreductase is an important index for evaluating the fluorescent probe, and the NO is tested by adopting the test solution with the same concentration₂-1 fluorescence intensity of different anions and cations and biological components, finding NO₂-1 shows a strong fluorescence change only under the combined action of nitroreductase and NADH. Also explored NO₂-1 detection of nitroreductase in hypoxic HeLa cells and normoxic HeLa cells, since during cellular hypoxia the intracellular nitroreductase content changes significantly, which affects the change in fluorescence intensity of the probe.

Drawings

FIG. 1 shows probe NO₂-1 reaction mechanism schematic in response to nitroreductase.

FIG. 2 shows probe NO₂-1 ultraviolet absorption spectrum verification pattern in response to nitroreductase.

FIG. 3 shows probe NO₂-1 fluorescence emission spectrum verification in response to nitroreductase.

FIG. 4 shows probe NO₂1 fluorescence spectra of nitroreductase solutions at different concentrations (a) and fluorescence intensity vs. NTR concentration (b) and (c)And a linear relationship diagram.

FIG. 5 shows probe NO₂Reaction time profiles of 1 in (a) nitroreductase test solutions of different concentrations (0. mu.g/mL, 0.5. mu.g/mL, 1. mu.g/mL and 3. mu.g/mL), (b) Probe NO₂-1 fluorescence spectra of different anions and cations and biological components.

FIG. 6 shows probe NO₂-1 two-photon absorption cross-sections in solution before and after reaction with nitroreductase.

FIG. 7 shows probe NO₂-1 cytotoxicity test chart.

FIG. 8 shows probe NO₂Confocal fluorescence imaging of HeLa cells co-stained simultaneously with 1 (10. mu.M) and 0.5. mu.M commercial mitochondrial probe (Mito-Tracker Green FM). Exploring NO₂-1 mitochondrial targeting ability.

FIG. 9 shows probe NO₂-1 confocal imaging of stained HeLa cells. (a) Is shown as NO₂-1 staining of HeLa cells under normoxia, (b) scheme NO₂-1 staining of HeLa cells under hypoxic conditions

Detailed Description

The invention is further illustrated by the following examples.

Example 1: NO₂Synthesis of (E) -1

At 45 deg.C, OH-1(1g, 2.53mmol), Cs₂CO₃(1.5g, 3.06mmol) and 4-nitrobenzylborobenzene (0.82g, 3.8mmol) were placed in a Schlenk flask under N₂Under an atmosphere, 10ml of DMF was added dropwise. Stirring at 45 deg.C for 4h, cooling to remove solvent, vacuum filtering with petroleum ether, purifying the obtained solid by column chromatography (dichloromethane: petroleum ether: 2:1), distilling under reduced pressure, and vacuum drying to obtain light yellow powder NO₂10.6 g, yield 64%.¹H NMR(400MHz,DMSO-d₆)δ8.26(d,2H)8.23(d,2H),8.13(d,2H),8.03(d,2H),7.86(m,2H),7.71(t,2H),7.21(d,2H),5.39(s,2H),4.03(s,3H),¹³C NMR(100MHz,CDCl₃)δ171.92,161.34,148.31,147.01,144.2,141.89,132.03,129.18,128.32,127.50,127.23,124.09,123.56,116.63,115.54,111.54,68.31,36.24.ESI-MS:m/z 403.1099([M-I]^-calcd 403.3842)。

Example 2: NO₂Spectroscopic test of-1

0.0053g of NO₂-1 was added to 5ml of DMSO to make a 2X 10 concentration^-3mol/L of test stock and 15. mu.L of the stock was pipetted into nitroreductase solutions of different concentrations (0.125. mu.g/ml, 0.25. mu.g/ml, 0.5. mu.g/ml, 1. mu.g/ml, 2. mu.g/ml, 3. mu.g/ml, 4. mu.g/ml, 5. mu.g/ml, 7. mu.g/ml). Subsequently, the fluorescence test was also performed on the above test, and as the concentration of nitroreductase was increased from 0.125. mu.g/mL to 7. mu.g/mL, the fluorescence intensity of the probe was also increased continuously (FIG. 4a), and after 3. mu.g/mL, the increase in the fluorescence intensity of the probe was small, and the nitroreductase concentration had a good linear relationship with the maximum fluorescence intensity of the probe (R) as shown in FIGS. 4b and 4c (R)²＝0.98)。

Example 3: NO₂Reaction mechanism test of-1

To study NO₂-1 reaction mechanism (FIG. 1), followed by NO₂-1 performing a UV test. As shown in FIG. 2, NO₂-1 shows a UV absorption peak around 400nm, a new UV absorption peak around 500nm is found after the nitroreductase and NADH are added, and the position of the UV absorption peak is the same as that of OH-1, and NO can be seen from the UV absorption spectrum₂-1 is reduced to OH-1 by nitroreductase and NADH. Subsequent verification from fluorescence spectra, NO₂-1 response mechanism. The probe NO after the reaction in the presence of nitroreductase, in the presence of NAHD and in the presence of nitroreductase and NADH, respectively, was tested₂-1 change in fluorescence intensity. As shown in FIG. 3, NO₂-1 shows a weak fluorescence signal after reaction with nitroreductase, whereas the probe NO₂-1 shows little fluorescence after interaction with NADH, but the probe NO is under the influence of NADH₂1A strong fluorescence signal around 558nm after the reaction with nitroreductase and an increase of more than 80 times in the fluorescence intensity of the probe compared to the absence of NADH, which is due to the energy supplied by NADH during the reaction, it can also be seen that probe NO is present₂-1 dependence of the reaction with nitroreductase on NADH.

Example 4: cytotoxicity test

At probe NO₂-1 Prior to the application of cellular imaging, the toxicity of the probe needs to be tested and the MTT method used for the experiment. Adding 0. mu.M, 5. mu.M, 10. mu.M and 20. mu.M probe NO, respectively₂-1 HeLa cells were cultured for 24h, and cell viability was consistently maintained above 85% (FIG. 7). Thus probe NO₂The-1 has low toxicity to HeLa cells and can be applied to biology.

Example 5: NO₂-1 sensitivity and interference tests

To study NO₂1 time to respond to nitroreductase, we performed real-time fluorescence tests on the probes in solutions of the responsive nitroreductase at concentrations of 0.5. mu.g/mL, 1. mu.g/mL, 3. mu.g/mL, respectively. As shown in FIG. 5a, the fluorescence intensity of the probe gradually increased with time, and nitroreductase and NO were reacted₂The reaction system of-1 reached a plateau at about 30min and NO₂-1 can be stably present in the system of the reaction. Subsequently to verify NO_2-Selectivity of 1-nitroreductase to prevent NO from other substances₂Interference of-1, we tested the addition of different anions and cations and biological components (Ca) to PBS buffer solution at PH 7.4²⁺、Cl^-、Mg²⁺、Na⁺、Cl^-、SO₄ ^2-、ClO^-、SO₃ ^2-、K⁺、HSO₃ ^-、Cys、GSH、H₂O₂、Glucose、S^2-NADH), as shown in fig. 5b, these interfering substances do not cause NO₂-1 change in fluorescence intensity.

Example 6: NO₂Two-photon Performance test of-1

Probe NO₂-1 in PBS solvent, no significant two-photon absorption. When the probe NO₂The effective two-photon absorption cross-section (. PHI.delta.) of-1 after reaction with nitroreductase and NADH appeared to be maximum at 820nm, which was 82GM (FIG. 6). Demonstration of Probe No₂-1 has potential for two-photon confocal fluorescence imaging of cellular intramitochondrial nitroreductase.

Example 7: cell localization assay

To study NO₂Mitochondrial localization Properties of-1, where mitochondrial commercial dyes (Mito-Tracker Green FM, 0.5. mu.M) with NO were used₂-1 Co-localization studies in HeLa cells were performed. The results show that NO₂Red channel of-1 (λ)_em＝500nm，λ_ex560nm) and Mito-Tracker Green FM (. lamda.) (M._em＝510±20nm，λ_ex480nm) fluorescence images overlap well and NO₂Pearson co-localization coefficient of-1 with mitochondrial commercial dye (Mito-Tracker Green FM) was calculated to be 0.91 (FIG. 8). These results show that NO₂-1 can be well localized in the mitochondria of living cells.

Example 8: NO₂-1 detection of nitroreductase to detect cellular hypoxia

To study NO₂-1 ability to detect nitroreductase in cells, we convert NO₂-1(10 μ M) was added to 2 groups of HeLa cells, one group was cultured with NO in an anoxic environment for 0.5h₂-1 culturing, another group being under normoxic atmosphere with NO₂-1 culturing. Then, confocal imaging is carried out by using an excitation light source with the wavelength of 500nm, and the fluorescence intensity is detected. As shown in FIG. 9, NO₂-1 emits weak red fluorescence upon incubation with HeLa cells under normoxic conditions and strong red fluorescence upon incubation with HeLa cells under hypoxic conditions. Shows that the level of nitroreductase of the HeLa cell is increased along with the reduction of the oxygen content, the fluorescence intensity of the probe is enhanced, and the probe NO is shown₂-1 Nitroreductases in HeLa cells can be detected and NO₂-1 has the potential to test whether a cell is hypoxic by detecting nitroreductase.