阅读说明：本技术 基于含氮杂环的中性线粒体荧光标记物及其制备方法与应用 (Neutral mitochondrial fluorescent marker based on nitrogen-containing heterocycle and preparation method and application thereof ) 是由 葛健锋 王越 孙如 于 2020-02-25 设计创作，主要内容包括：本发明公开了基于含氮杂环的中性线粒体荧光标记物及其制备方法与应用,本发明首次公开的中性荧光团为含有N-H键的杂环,靶向线粒体解决了现有中性结构的荧光染料细胞器靶向能力是随机的且不确定的问题,也避免中性荧光团是细胞中脂滴的商业标记物的问题,本发明改善荧光团良好的光学性能的同时,通过对其结构创造性的修饰来调控原荧光团的细胞器靶向能力,而且标记物改善荧光团的生物学性能,含氮杂环砌块廉价易得,有利于控制新染料的成本,具有重大的科学意义和商业价值。(The invention discloses a neutral mitochondrial fluorescent marker based on nitrogen-containing heterocycle and a preparation method and application thereof, the neutral fluorophore disclosed for the first time is a heterocycle containing N-H bonds, the targeted mitochondria solves the problems that the cellular organelle targeting capability of the existing fluorescent dye with a neutral structure is random and uncertain, and the neutral fluorophore is a commercial marker of lipid droplets in cells.)

1. The neutral mitochondrial fluorescent marker based on the nitrogen-containing heterocycle is characterized by being one of the following chemical structural formulas:

wherein, X¹、X²Independently selected from CH or a heteroatom; m, E, E¹、B₁Independently selected from alkyl groups having less than 6 carbon atoms.

2. The nitrogen-heterocycle based neutral mitochondrial fluorescent marker of claim 1, wherein the nitrogen-heterocycle based neutral mitochondrial fluorescent marker is one of the following chemical formulas:

X¹selected from CH or N; x²Selected from CH or N.

3. Use of the neutral mitochondrial fluorescent marker based on nitrogen-containing heterocycles according to claim 1 for mitochondrial fluorescent labeling; or the use of the neutral mitochondrial fluorescent marker based on nitrogen-containing heterocycles according to claim 1 for preparing a mitochondrial fluorescent labeling reagent.

4. The method for preparing neutral mitochondrial fluorescent marker based on nitrogen-containing heterocycle according to claim 1, which is one of the following methods:

(1) reacting the compound 6 with the compound 7 to obtain a compound 8; deprotecting the compound 8 to obtain a neutral mitochondrial fluorescent marker based on a nitrogen-containing heterocycle;

(2) reacting the compound 9 with the compound 7 to obtain a compound 10; deprotecting the compound 10 to obtain a neutral mitochondrial fluorescent marker based on a nitrogen-containing heterocycle;

(3) reacting the compound 13 with the compound 7 to obtain a compound 14; and (3) deprotecting the compound 14 to obtain the neutral mitochondrial fluorescent marker based on the nitrogen-containing heterocycle.

5. The method for preparing neutral mitochondrial fluorescence marker based on nitrogen-containing heterocycle according to claim 4, wherein deprotection is performed in the presence of hydrochloric acid; the reaction of the compound 6 with the compound 7 is carried out in the presence of a noble metal salt catalyst; the reaction of the compound 9 with the compound 7 is carried out in the presence of a noble metal salt catalyst; the reaction of the compound 13 with the compound 7 is carried out in the presence of a noble metal salt catalyst.

6. The method of claim 5, wherein the noble metal salt catalyst comprises palladium salt catalyst.

7. A method of imaging cells, comprising the steps of:

(1) reacting the compound 6 with the compound 7 to obtain a compound 8; deprotecting the compound 8 to obtain a neutral mitochondrial fluorescent marker based on a nitrogen-containing heterocycle;

(2) reacting the compound 9 with the compound 7 to obtain a compound 10; deprotecting the compound 10 to obtain a neutral mitochondrial fluorescent marker based on a nitrogen-containing heterocycle;

(3) reacting the compound 13 with the compound 7 to obtain a compound 14; deprotection of the compound 14 to obtain a neutral mitochondrial fluorescent marker based on a nitrogen-containing heterocycle;

(4) co-culturing the neutral mitochondrial fluorescent marker prepared in the step (1) or the step (2) based on the nitrogen-containing heterocycle and cells, adding a mitochondrial red marker, continuing culturing, and performing cell imaging;

or co-culturing the neutral mitochondrial fluorescent marker based on the nitrogen-containing heterocycle prepared in the step (3) and cells, adding a mitochondrial green marker, continuing culturing, and then imaging the cells.

8. The cell imaging method according to claim 7, wherein the deprotection is carried out in the presence of hydrochloric acid; the reaction of the compound 6 with the compound 7 is carried out in the presence of a noble metal salt catalyst; the reaction of the compound 9 with the compound 7 is carried out in the presence of a noble metal salt catalyst; the reaction of the compound 13 with the compound 7 is carried out in the presence of a noble metal salt catalyst.

9. The method of claim 7, wherein the imaging of the cells is performed by confocal laser microscopy; exciting a blue light channel by using 405nm, and collecting a fluorescence signal within a range of 410-500 nm; exciting a red light channel by using 561nm, and collecting a fluorescence signal within the range of 570-750 nm; and (3) exciting the green light channel by using 488 nm, and collecting a fluorescence signal within the range of 500-550 nm.

10. The method of claim 7, wherein the cells comprise normal cells, cancer cells.

Technical Field

The invention belongs to a fluorescence labeling technology, and particularly relates to a novel neutral mitochondrial fluorescence label based on a nitrogen-containing heterocycle.

Background

Mitochondria are one of the most basic organelles in cells, and are involved in important physiological activities such as Cell genetic material transfer and Cell differentiation in addition to providing energy to cells as a main site of aerobic respiration (see: L evenson, r.; Macara, i. g.; Smith, r. L.; Cantley, L.; Housman, d. Cell 1982, 28, 855.), etc. thus, it is important to monitor mitochondria in real time in scientific research.Angew Chem Int Ed2016,55,13658.). Even the most commonly used commercial mitochondrial red and green markers. This is because the presence of a proton pump on the inner mitochondrial membrane makes it easier for these cationic dyes to penetrate the mitochondrial membrane and accumulate in the mitochondria. The problem is also compounded by the fact that entry of these cations into mitochondria changes the mitochondrial membrane potential, causing apoptosis (see:Sens Actuators B2019,292, 16.）。

disclosure of Invention

The invention discloses a novel neutral mitochondrial fluorescent marker based on nitrogen-containing heterocycle, which can be used as a mitochondrial fluorescent marker, solves the problems that the organelle targeting ability of the existing fluorescent dye with a neutral structure is random and uncertain for the first time, and avoids the problem that a neutral fluorophore is a commercial marker of lipid droplets in cells.

The invention adopts the following technical scheme:

the neutral mitochondrial fluorescent marker based on the nitrogen-containing heterocycle is one of the following chemical formulas:

、、

wherein, X¹、X²Independently selected from CH or a heteroatom; m, E, E¹、B₁Independently selected from alkyl with the carbon number less than 6; the neutral mitochondrial fluorescent marker based on the nitrogen-containing heterocycle contains an N-H bond.

Preferably, the neutral mitochondrial fluorescent marker based on the nitrogen-containing heterocycle is one of the following chemical formulas:

X¹selected from CH or N; x²Selected from CH or N.

The invention discloses the application of the neutral mitochondrial fluorescent marker based on the nitrogen-containing heterocycle in mitochondrial fluorescent marking; or the application of the neutral mitochondrial fluorescence marker based on the nitrogen-containing heterocycle in preparing a mitochondrial fluorescence marker reagent.

The invention discloses a preparation method of the neutral mitochondrial fluorescent marker based on the nitrogen-containing heterocycle, which is characterized by comprising the following steps:

(1) reacting the compound 6 with the compound 7 to obtain a compound 8; deprotecting the compound 8 to obtain a neutral mitochondrial fluorescent marker based on a nitrogen-containing heterocycle;

(2) reacting the compound 9 with the compound 7 to obtain a compound 10; deprotecting the compound 10 to obtain a neutral mitochondrial fluorescent marker based on a nitrogen-containing heterocycle;

(3) reacting the compound 13 with the compound 7 to obtain a compound 14; and (3) deprotecting the compound 14 to obtain the neutral mitochondrial fluorescent marker based on the nitrogen-containing heterocycle.

The invention discloses a cell imaging method, which comprises the following steps:

(1) reacting the compound 6 with the compound 7 to obtain a compound 8; deprotecting the compound 8 to obtain a neutral mitochondrial fluorescent marker based on a nitrogen-containing heterocycle;

(2) reacting the compound 9 with the compound 7 to obtain a compound 10; deprotecting the compound 10 to obtain a neutral mitochondrial fluorescent marker based on a nitrogen-containing heterocycle;

(3) reacting the compound 13 with the compound 7 to obtain a compound 14; deprotection of the compound 14 to obtain a neutral mitochondrial fluorescent marker based on a nitrogen-containing heterocycle;

(4) co-culturing the neutral mitochondrial fluorescent marker prepared in the step (1) or the step (2) based on the nitrogen-containing heterocycle and cells, adding a mitochondrial red marker, continuing culturing, and performing cell imaging;

or co-culturing the neutral mitochondrial fluorescent marker based on the nitrogen-containing heterocycle prepared in the step (3) and cells, adding a mitochondrial green marker, continuing culturing, and then imaging the cells. The cells include normal cells and cancer cells.

In the present invention, deprotection is carried out in the presence of hydrochloric acid; the reaction of compound 6 with compound 7 is carried out in the presence of a noble metal salt catalyst, preferably under basic conditions; the reaction of the compound 9 with the compound 7 is carried out in the presence of a noble metal salt catalyst, preferably under basic conditions; the reaction of compound 13 with compound 7 is carried out in the presence of a noble metal salt catalyst, preferably under basic conditions. Preferably, the noble metal salt catalyst comprises a palladium salt catalyst.

In the present invention, the chemical structural formula of the compound is as follows:

the chemical structure of compound 14 is as follows:

wherein the heterocyclic rings contain an N-H bond, X¹、X²Independently selected from CH or a heteroatom; m, E, E¹、B₁Is a substituent and is independently selected from alkyl with the carbon number less than 6. The alkyl group in the present invention represents a saturated branched or straight chain monovalent hydrocarbon group having 1 to 6 carbon atoms, such as methyl (Me), n-butyl (Bu), ethyl (Et), and the like.

In the invention, a laser confocal microscope is used for cell imaging; exciting a blue light channel by using 405nm, and collecting a fluorescence signal within a range of 410-500 nm; exciting a red light channel by using 561nm, and collecting a fluorescence signal within the range of 570-750 nm; and (3) exciting the green light channel by using 488 nm, and collecting a fluorescence signal within the range of 500-550 nm.

The invention discloses a nitrogen heterocycle-based neutral mitochondrial fluorescent marker for cellular neutral mitochondrial fluorescent labeling for the first time, and cell imaging can be realized after the marker is co-cultured with cells. The invention improves the good optical performance of the fluorophore, regulates the organelle targeting ability of the original fluorophore by creatively modifying the structure of the fluorophore, has low cytotoxicity during cell imaging, little damage to a biological sample, no influence of other organelles, can observe the cell sample for a long time, improves the biological performance of the fluorophore by the marker, has cheap and easily obtained nitrogenous heterocyclic building blocks, and is beneficial to controlling the cost of a new dye.

Drawings

FIG. 1 is a scheme for the synthesis of dyes to which the present invention relates;

FIG. 2 is a NMR spectrum of dye 1 a;

FIG. 3 shows the UV-VIS absorption spectrum and fluorescence spectrum of dye 1a in chloroform;

FIG. 4 shows the UV-VIS absorption spectrum and the fluorescence spectrum of dye 1b in chloroform;

FIG. 5 shows the UV-VIS absorption spectrum and fluorescence spectrum of dye 1c in chloroform;

FIG. 6 is a UV-VIS absorption spectrum and a fluorescence spectrum of dye 2a in chloroform;

FIG. 7 is a UV-VIS absorption spectrum and a fluorescence spectrum of dye 2b in chloroform;

FIG. 8 is a UV-VIS absorption spectrum and a fluorescence spectrum of dye 2c in chloroform;

FIG. 9 is a UV-VIS absorption spectrum and a fluorescence spectrum of dye 3a in chloroform;

FIG. 10 is a UV-VIS absorption spectrum and a fluorescence spectrum of dye 3b in chloroform;

FIG. 11 is a UV-VIS absorption spectrum and a fluorescence spectrum of dye 3c in chloroform;

FIG. 12 is a UV-VIS absorption spectrum and a fluorescence spectrum of dye 3d in chloroform;

FIG. 13 is a UV-VIS absorption spectrum and a fluorescence spectrum of dye 4 in chloroform;

FIG. 14 is an image of dye 1a in L929 cells and He L a cells;

FIG. 15 is an image of dye 1b in L929 cells and He L a cells;

FIG. 16 is an image of dye 1c in L929 cells and He L a cells;

FIG. 17 is an image of dye 2a in L929 cells and He L a cells;

FIG. 18 is an image of dye 2b in L929 cells and He L a cells;

FIG. 19 is an image of dye 2c in L929 cells and He L a cells;

FIG. 20 is an image of dye 3a in L929 cells and He L a cells;

FIG. 21 is an image of dye 3b in L929 cells and He L a cells;

FIG. 22 is an image of dye 3c in L929 cells and He L a cells;

FIG. 23 is an image of dye 3d in L929 cells;

FIG. 24 is an image of dye 3d in He L a cells;

fig. 25 is an image of dye 4 in He L a cells.

Detailed Description

The synthetic route of the embodiment of the invention is shown in figure 1, and the lower number of the chemical formula represents a compound. In the synthesis of the compound, the raw material proportion and the purification method adopt the conventional proportion or the conventional purification method, and the examples are schematically expressed.