A Flavone-Based Long-Wavelength Fluorescent Probe to Detect Biothiols in vitro and in vivo

doi:10.6023/cjoc202206016

Abstract

Biothiols, such as cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) play crucial roles in physiological and pathological processes. GSH, especially, is overexpressed in numerous types of tumor cells and could be considered as a biomarker. However, owing to the difficulty and challenge in sensing normal and cancer cells simultaneously, it is important to solve this problem in biological processes. Herein, a naked-eye visible, “off-on” long-wavelength fluorescent probe (4) based on N,N-(dimethylamino)naphthalenyl flavone (3) was designed and synthesized. This probe showed high selectivity for biothiols and low detection limits. The mechanism study revealed that biothiols catalyzed the cleavage of the ester bond, and strongly fluorescent flavone 3 was released. Probe 4 could be used for the imaging of biothiols in living cells and mice. In addition, the probe was optimized to achieve the selective imaging of normal hepatocyte HL-7702 and heptocarcinoma cell HepG2.

Key words: long-wavelength, fluorescence probe, flavone, biothiol

Cite this article

Siyi Zhou, Xu Ding, Yongmei Zhao, Jinghua Li, Wen Luo. A Flavone-Based Long-Wavelength Fluorescent Probe to Detect Biothiols in vitro and in vivo[J]. Chinese Journal of Organic Chemistry, 2023, 43(1): 178-185.

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Fig. & Tab. 9

Scheme 1. Synthetic route of probe 4 Reagents and conditions: (a) ClCH2CN, HCl, Et2O, 0 ℃; (b) HCl, H2O, reflux; (c) 6-(dimethylamino)-2-naphthaldehyde, 10% NaOH, EtOH, r.t.; (d) DBS chloride, Et3N, CHCl3, 0 ℃

Figure 1. Absorption spectra (A) and fluorescence spectra (B) of 4 (10 μmol/L) upon the addition of amino acids or biothiols (20 μmol/L) in a THF-water solution (V∶V=3∶7, 10 mmol/L PBS, pH 7.4)

Figure 2. Photographs of probe 4 (10 μmol/L) with visual color changes upon the addition of different amino acids or GSH (20 μmol/L) in a THF-water solution (V∶V=3∶7, 10 mmol/L PBS, pH 7.4)

Figure 3. Fluorescent changes of probe 4 (10 μmol/L) in the presence of various concentration of Cys in a THF-water solution (V∶V=3∶7, 10 mmol/L PBS, pH 7.4) solution at room temperature. The excitation wavelength was 452 nm. The incubation time was 25 min

Figure 4. Sensing mechanism of probe 4

Figure 5. Cell viability of three cell lines after treatments with probe 4 (a) or flavone 3 (b) for 24 h. Results are expressed as the mean ±S.D (n=3)

Figure 6. Confocal images of living Hela cells. Hela cells were co-stained with probe 4 (10 μmol/L, A1~E1) and DAPI (1 μg/mL, A2~E2). Merged photographs (A3~E3) are at bottom. λex=405 and 452 nm for DAPI and probe 4, respectively

Figure 7. Confocal images of co-incubation of HepG2 and HL-7702 cells after treated with probe 4 (10 μmol/L, a~d) or flavone 3 (5 μmol/L, e~h). (a, e) For DAPI, λex=405 nm, λem=414~442 nm. (b,f) For mitochondrial dye, λex=577 nm, λem=590~620 nm (shown in green, pseudo color). (c, g) For 4 or 3, λex=452 nm, λem=630~660 nm. (d, h) Merged imgaes

Figure 8. (a) Fluorescence images of BALB/c nude mice that was subcutaneously injected with glucose (50 μL, left rear side) or probe 4 [50 μL, 200 μmol/L in DMSO/glucose (V∶V=1∶9), right rear side]; (b) relative fluorescence intensity at different times of Figure 8(a). (c) fluorescence of biothiols in BALB/c nude mice that were intraperitoneally injected with or without NEM and then treated by probe 4 [50 μL, 200 μmol/L in DMSO/glucose (V∶V=1∶9)] (a) Images were captured at 0, 1, 3, 6, 9 and 28 min. (b) Images were captured at 0 and 10 min]. λex=460 nm, λem=600~640 nm

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