Use of a heterocyclic compound as a fluorescent probe

Abstract

The present application relates to a kind of heterocyclic compounds in the application as fluorescent probe.The structure formula of the heterocyclic compound is as shown in general formula I), wherein, R1, R2, R3, R4, R5, R6, R7 And R8 Independently hydrogen, alkyl, hydroxyl, phenyl, hydroxyphenyl, indole, oxymethyl one of them.The present application finds that the heterocyclic compound as shown in general formula I) can be specifically located in mitochondria, and these heterocyclic compounds have longer fluorescence lifetime, therefore, these heterocyclic compounds are more suitable for use as mitochondrial fluorescent probe.General formula I).

Description

Technical Field

[0001] This invention relates to the field of heterocyclic compounds, and particularly to the application of a heterocyclic compound as a fluorescent probe. Background Technology

[0002] Mitochondria, as the core organelle of cellular energy metabolism, are closely related to cell function, disease occurrence, and drug action mechanisms through their dynamic changes. Developing efficient and precise mitochondrial indicators is of great significance for cell biology research, disease diagnosis, and drug screening. Currently, fluorescent probes are the main tool for studying mitochondrial function, but existing mitochondrial fluorescent probes have significant drawbacks: 1) Limited fluorescence lifetime and photostability: Short fluorescence lifetime (nanoseconds), susceptible to photobleaching, and a significant decrease in signal-to-noise ratio during long-term imaging, making it difficult to meet the needs of long-term, high-resolution dynamic monitoring; 2) Limited targeting specificity: Some probes have insufficient localization specificity and are prone to cross-labeling with other organelles (such as lysosomes and endoplasmic reticulum) (e.g., Rhodamine 123); 3) Environmental sensitivity and signal interference: Some probes are easily affected by the mitochondrial microenvironment (pH, ROS, ion concentration), requiring complex corrections (e.g., GCaMP series is affected by pH fluctuations, MitoSOX signal depends on membrane potential), reducing detection accuracy; 4) Insufficient compatibility with multicolor imaging: Most existing mitochondrial probes emit spectra concentrated in green (e.g., Rhodamine 123, 530). The wavelengths of mitochondria (nm) or orange-red bands show significant spectral overlap with commonly used fluorescent labels such as green fluorescent protein (GFP), red fluorescent protein (mCherry), and blue fluorescent DNA dye (4′,6-diamidindo-2-phenylindole, DAPI), leading to crosstalk of multi-parameter labeled signals and limiting the synchronous analysis of mitochondria with other organelles or signaling pathways. While a few blue fluorescent probes can avoid spectral conflicts, they require dedicated UV excitation equipment, limiting their applicability and making it difficult to meet the flexibility requirements of high-throughput imaging for simultaneous multi-parameter detection. 5) Cytotoxicity and functional impact: Some probes (such as DiOC6) are toxic due to inhibiting enzyme activity or disrupting membrane structure, and long-term use affects cell viability and mitochondrial metabolism. Although studies have attempted to optimize probe performance by enhancing fluorescence intensity or extending lifetime, these improvements often come at the cost of sacrificing localization specificity or increasing synthesis difficulty.

[0003] In summary, fluorescent probes need to have excellent resistance to photobleaching in long-term live-cell imaging; in multiple labeling experiments, the emission spectrum of fluorescent probes needs to be compatible with existing dyes; and in pathological mechanism studies, fluorescent probes need to be highly sensitive to changes in mitochondrial membrane potential. Summary of the Invention

[0004] One aspect of the present invention provides a heterocyclic compound with the structural formula shown in general formula I).

[0005] General Formula I).

[0006] Among them, R1, R2, R3, R4, R5, R6, R7 and R8 are independently one of hydrogen, alkyl, hydroxyl, phenyl, hydroxyphenyl, indole and oxymethyl.

[0007] In one specific embodiment, the alkyl group is one of methyl, ethyl, and propyl.

[0008] In one specific embodiment, R1 and R6 are independently hydrogen or hydroxyl groups.

[0009] In one specific embodiment, R2, R3, R4, and R5 are independently hydrogen or alkyl groups.

[0010] In one specific embodiment, R7 is one of phenyl, indole, and hydroxyphenyl.

[0011] In one specific embodiment, R8 is one of hydrogen, indole, and oxymethyl.

[0012] In one specific embodiment, the heterocyclic compound is at least one of oligosporin A, oligosporin B, oligosporin C, oligosporin D, oligosporin E, oligosporin F, oligosporin G, oligosporin J, oligosporin H, oligosporin I, oligosporin K, and oligosporin L.

[0013] The second invention provides the application of the heterocyclic compound according to any one of the inventions as a fluorescent probe.

[0014] In one specific embodiment, the application is as a mitochondrial fluorescent probe.

[0015] The beneficial effects of the present invention are as follows: The present invention discovers that heterocyclic compounds as shown in general structural formula I) can be specifically localized to mitochondria, and these heterocyclic compounds have a longer fluorescence lifetime. Therefore, these heterocyclic compounds are more suitable as mitochondrial fluorescent probes. Attached Figure Description

[0016] Figure 1 Oligosporin B, oligosporin K, and oligosporin L are shown to be localized on mitochondria in human embryonic kidney cell line HEK-293T, human glioblastoma cell line U87, and human non-small cell lung cancer cell line A549. Blue: nuclear dye DAPI; green: mitochondrial dye MitoTracker. TM green FM.

[0017] Figure 2The fluorescence absorption spectrum, emission spectrum, and fluorescence lifetime plot of the heterocyclic compound oligosporin B in the kidney cell line HEK-293T are shown in MitoTracker. TM Red FM: Red mitochondrial dye as a positive control.

[0018] Figure 3 The average fluorescence lifetime histogram (left) and fluorescence lifetime spectrum (right) of oligosporin B, oligosporin K, and oligosporin L are shown. Detailed Implementation

[0019] The present invention will be further described in detail below through preferred embodiments, but these embodiments do not constitute a limitation thereof.

[0020] Unless otherwise specified, the reagents and other materials used in the embodiments of this invention can be purchased commercially.

[0021] For details on the synthesis of the heterocyclic compounds oligosporins A, B, C, D, E, F, G, and J, please refer to "Unexpected biosynthesis of fluorescein-like arthrocolins against resistant strains in an engineered..." Escherichia coli .Org. Lett. 2019, 21, 6499−6503.

[0022] For details on the synthesis of the heterocyclic compounds oligosporin H, oligosporin I, oligosporin K, and oligosporin L, please refer to Harnessing in vivo synthesis of bioactive multiarylmethanes in Escherichia coli viaoxygen-mediated free radical reaction induced by simple phenols. Microb CellFact. 2024, 23, 219-231.

[0023] The structures of the heterocyclic compounds oligosporins A, B, C, D, E, F, G, J, H, I, K, and L are shown in general formula I).

[0024] General Formula I).

[0025] When R1 and R6 are hydroxyl groups, R2 and R5 are methyl groups, R3 and R4 are hydrogen groups, R7 is phenyl, and R8 is indole, the heterocyclic compound is oligosporin A; when R1 and R6 are hydroxyl groups, R2 and R4 are methyl groups, R3 and R5 are hydrogen groups, R7 is phenyl, and R8 is indole, the heterocyclic compound is oligosporin B; when R1 and R6 are hydroxyl groups, R3 and R4 are methyl groups, R2 and R5 are hydrogen groups, R7 is phenyl, and R8 is indole, the heterocyclic compound is oligosporin C; the molecular formula of oligosporin A, oligosporin B, and oligosporin C is C0. 29 H 23 NO3, with a molecular weight of 433, is shown in Formula II).

[0026] Formula II).

[0027] When R1 and R6 are hydroxyl groups, R2, R3, R4, and R5 are hydrogen, R7 is phenyl, and R8 is indole, the heterocyclic compound is oligosporin D; the molecular formula of oligosporin D is C1. 27 H 19 NO3, with a molecular weight of 405, is shown in Formula III.

[0028] Formula III).

[0029] When R1 and R6 are hydroxyl groups, R2, R3, R4, and R5 are hydrogen, R7 is phenyl, and R8 is oxymethyl, the heterocyclic compound is oligosporin E; the molecular formula of oligosporin E is C1. 20 H 16 O4, with a molecular weight of 320, is shown in Formula IV.

[0030] Formula IV).

[0031] When R1 and R6 are hydroxyl groups, R2, R3, R4, R5, and R8 are hydrogen atoms, and R7 is a phenyl group, the heterocyclic compound is oligosporin F; the molecular formula of oligosporin F is C1. 19 H 14 O3, with a molecular weight of 290, is shown in formula V.

[0032] Formula V).

[0033] When R1 and R6 are hydroxyl groups, R2 and R4 are methyl groups, R3, R5, and R8 are hydrogen, and R7 is phenyl, the heterocyclic compound is oligosporin G; when R1 and R6 are hydroxyl groups, R3 and R4 are methyl groups, R2, R5, and R8 are hydrogen, and R7 is phenyl, the heterocyclic compound is oligosporin J; the molecular formulas of both oligosporin G and oligosporin J are C1. 21 H 18 O3, with a molecular weight of 318, is shown in Formula VI.

[0034] Formula VI).

[0035] When R1 and R6 are hydroxyl groups, R2 and R5 are methyl groups, R3 and R4 are hydrogen groups, R7 is hydroxyphenyl, and R8 is indole, the heterocyclic compound is oligosporin H; when R1 and R6 are hydroxyl groups, R2 and R4 are methyl groups, R3 and R5 are hydrogen groups, R7 is hydroxyphenyl, and R8 is indole, the heterocyclic compound is oligosporin I; the molecular formulas of both oligosporin H and oligosporin I are C1. 29 H 23 NO4 has a molecular weight of 449, as shown in Formula VII).

[0036] Equation VII).

[0037] When R1, R2, R3, R4, R5, R6, and R8 are hydrogen, and R7 is indole, the heterocyclic compound is oligosporin K; the molecular formula of oligosporin K is C1. 21 H 15 NO, with a molecular weight of 297, as shown in Formula VIII.

[0038] Formula VIII).

[0039] When R1, R2, R3, R4, R5, and R6 are hydrogen, and R7 and R8 are indole, the heterocyclic compound is oligosporin L; the molecular formula of oligosporin L is C1. 29 H 20 N₂O, with a molecular weight of 412, is shown in Formula IX.

[0040] Formula IX).

[0041] The renal cell line HEK-293T, the human glioblastoma cell line U87, and the human non-small cell lung cancer cell line A549 were all purchased from Wuhan Pronosei Life Science Technology Co., Ltd.

[0042] Using dimethyl sulfoxide (DMSO) as a solvent, heterocyclic compounds oligosporin B, oligosporin K, oligosporin L, and the green fluorescent probe MitoTracker were combined. TM Green FM (purchased from Thermo Fisher Scientific), 4',6-diamidinyl-2-phenylindole (DAPI), and the red fluorescent probe MitoTracker TM Red FM (purchased from Thermo Fisher Scientific) was prepared as a 200 μg / mL solution.

[0043] 100 μg of poly-L-ornithine and 10 μg of laminin were dissolved together in 1 ml of phosphate buffer (PBS, pH 7.4) to prepare a coating solution of 100 μg / mL poly-L-ornithine and 10 μg / mL laminin. Example 1

[0044] Confocal microscopy was used to observe the subcellular localization of oligosporin B, oligosporin K, and oligosporin L in the renal cell line HEK-293T, the human glioblastoma cell line U87, and the human non-small cell lung cancer cell line A549.

[0045] 100 μL of coating solution was added to the cell microculture slide chamber and solidified at 37°C for 2 hours to obtain coated slides. 100 μL of 4 × 10⁻⁶ cells / mL was then added to the slide chamber. 6 The number of kidney cell lines HEK-293T, human glioblastoma cell line U87, and human non-small cell lung cancer cell line A549 (4 × 10⁻⁶ cells / mL) were used. 5 Cells were seeded onto coated glass slides and incubated at 37°C for 24 h in a 5% CO2 incubator to allow cell colonization. Subsequently, 2 μL of oligosporin B, oligosporin K, and oligosporin L solutions were added to each slide to achieve a final concentration of 2 μg / mL for each heterocyclic compound. After incubation for 1 h, cells were washed three times with 500 μL of PBS to remove unbound heterocyclic compounds. Cells were then fixed with 1% paraformaldehyde fixative at room temperature for 1 h. After fixation, cells were washed three more times with 500 μL of PBS, followed by the addition of the mitochondrial-localized green fluorescent probe MitoTracker. TM Green FM was used to stain cells for mitochondria by incubation at 37°C for 30 min. Cells were then washed three times with 500 μL of PBS to remove unbound green fluorescent probes. Next, the cells were incubated with the nucleus-localized blue fluorescent probe 4',6-diamidinyl-2-phenylindole (DAPI) for 5 min to stain the nuclei. The cells were then washed three times with 500 μL of PBS to remove unbound blue fluorescent probes. The PBS solution was blotted dry, the slide chamber bottom plate was removed, a coverslip was placed on, and the slide was sealed with nail polish to obtain the test sample. The test sample was co-localized using a LEICA TCS SP8 confocal microscope to evaluate whether each heterocyclic compound was localized within the mitochondria. The results are as follows: Figure 1 As shown.

[0046] Figure 1 The results showed that the subcellular localization of oligosporin B, oligosporin K, and oligosporin L in the kidney cell line HEK-293T, the human glioblastoma cell line U87, and the human non-small cell lung cancer cell line A549 indicated that all three metabolites were located within the mitochondria of the cells. Example 2

[0047] Scanning of total emission and excitation spectra was used to measure the fluorescence lifetimes of oligosporin B, oligosporin K, and oligosporin L.

[0048] Add 100 μL of coating solution to the cell microculture slide chamber and incubate at 37°C for 2 hours. Add 100 μL of 4 × 10⁻⁶ cells / mL of the coating solution to the slide chamber. 6 HEK-293T renal cell line (4 × 10⁶ cells / mL) 5 Cells were seeded and coated onto glass slides, which were then incubated in a 5% CO2 incubator at 37°C for 24 h. 2 μL of oligosporin B solution was added to the cell microculture slide chamber to achieve a final oligosporin B concentration of 2 μg / mL. After incubation for 1 h, the cells were washed three times with 500 μL of PBS solution and fixed with 1% paraformaldehyde fixative at room temperature for 1 h. Subsequently, the cells were washed three times with 500 μL of PBS solution, the PBS solution was blotted dry, the bottom plate of the slide chamber was removed, a coverslip was placed on top, and the plate was sealed with nail polish to obtain the oligosporin B test sample. The total emission and excitation spectra of the test sample were measured using a LEICA TCS SP8 confocal microscope. The results are shown below. Figure 2 As shown.

[0049] Replace the aforementioned oligosporin B with the red fluorescent probe MitoTracker, which is localized to mitochondria. TM Red FM prepared positive control test samples, and the results were as follows: Figure 2 As shown.

[0050] Figure 2 The results showed that oligosporin B had an optimal excitation spectrum of 571 nm and an optimal emission spectrum of 611 nm, classifying it as a red fluorescent probe.

[0051] The above oligosporin B was replaced with oligosporin K and oligosporin L to prepare test samples of oligosporin K and oligosporin L, respectively.

[0052] Time-correlated single-photon counting was employed to measure the fluorescence lifetimes of oligosporin B, oligosporin K, and oligosporin L samples on a LEICA TCS SP8 confocal inverted microscope equipped with a disk anode microchannel plate photomultiplier tube (Europhoton, Berlin, Germany). This microscope allows for time- and spatially resolved photon detection. Spatial resolution can be achieved using a quadrant anode. The excitation source was a 555 nm picosecond diode laser with a single-pulse energy... Ep=2 nJ, system transmission efficiency of 60%, irradiation of a relatively large area with a diameter of approximately 100 micrometers, and operating frequency of 1 MHz. The estimated average excitation power of the excitation source is approximately 15.3 mW•cm. -2 (where the average power P) 平均 =Ep×f=2 nJ×1 MHz=2mW,P 测试杂环化合物 =P 平均 ×60%=1.2 mW; Spot area A=π (50 micrometers) 2 ≈7.85×10 -9 m 2 Average excitation power I 测试杂环化合物 =P 测试杂环化合物 ÷A = 1.2 mW ÷ 7.85 × 10 -9 m 2 ≈15.3mW / cm -2 This corresponds to the single-photon counting level. Fluorescence lifetime was measured using a FLIM mounted at 555 nm on a LEICA TCS SP8 confocal inverted microscope. Fluorescence lifetime data were analyzed using a global analysis of multi-exponential decay. The average fluorescence lifetime histograms and fluorescence lifetime spectra of the heterocyclic compounds oligosporin B, oligosporin K, and oligosporin L are shown below. Figure 3 As shown in Table 1, the solvent relaxation time fitting parameters for the heterocyclic compounds oligosporin B, oligosporin K, and oligosporin L at 555 nm in HEK-293T cells are shown in Table 1. The MitoTracker data at the 555 nm emission band are compared with those of the positive control. TM Compared to Red FM, the fluorescence lifetime of the heterocyclic compound oligosporin B (<τs>=3.12±0.2 ns) was approximately 1.71 times that of the positive control (<τs>=1.83±0.2 ns), while the fluorescence lifetimes of the heterocyclic compounds oligosporin K and oligosporin L were approximately 1.84 times and 1.03 times that of the positive control, respectively. This indicates that the heterocyclic compounds oligosporin B, oligosporin K, and oligosporin L have potential application value as fluorescent dyes.

[0053] Table 1

[0054]

[0055] Note: χ 2: Goodness-of-fit parameter, a statistical measure of the degree of fit between the experimentally measured fluorescence decay curve and the multi-exponential decay model used. A χ² value between 1.0 and 1.5 is generally considered a good fit; τ1: fast relaxation process, τ2: medium-speed relaxation process, τ3: slow relaxation process; τ1, τ2, τ3: fluorescence lifetime components obtained from the fit; α1, α2, α3: weighting factors corresponding to the fluorescence lifetime components (τ1, τ2, τ3), representing the contribution ratio of different lifetime components (τ1, τ2, τ3) to the overall fluorescence decay curve. It is a normalized value that satisfies α1+α2+α3=1; <τ> is the amplitude-weighted average lifetime, i.e., the fluorescence lifetime, <τ>=τ1(α1)+τ2(α2)+τ3(α3); [a]=±0.2 ns.

Claims

1. The application of a heterocyclic compound in the preparation of mitochondrial fluorescent probes, wherein the heterocyclic compound has the structural formula shown in general formula I). General Formula I). in, R1, R2, R3, R4, R5, R6 and R7 are independently one of hydrogen, alkyl, hydroxyl, phenyl, hydroxyphenyl and indole, and R8 is indole.

2. The application according to claim 1, characterized in that, The alkyl group is one of methyl, ethyl, and propyl.

3. The application according to claim 1, characterized in that, R1 and R6 are independently hydrogen or hydroxyl groups.

4. The application according to claim 1, characterized in that, R2, R3, R4, and R5 are independently hydrogen or alkyl groups.

5. The application according to claim 1, characterized in that, R7 is one of phenyl, indole, and hydroxyphenyl.

6. The application according to claim 1, characterized in that, The heterocyclic compound is at least one of oligosporin A, oligosporin B, oligosporin C, oligosporin D, oligosporin H, oligosporin I, oligosporin K, and oligosporin L, wherein the structural formulas of oligosporin A, oligosporin B, and oligosporin C are shown in Formula II), the structural formula of oligosporin D is shown in Formula III), the structural formulas of oligosporin H and oligosporin I are shown in Formula VII), the structural formula of oligosporin K is shown in Formula VIII), and the structural formula of oligosporin L is shown in Formula IX). Formula II) Formula III) Equation VII) Formula VIII). Formula IX).

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