A Novel Phosphate-Ester-Phenothiazine Fluorescent Probe for Detection and Imaging of Sulfur Dioxide Derivatives

doi:10.6023/cjoc202603043

Abstract

A novel phenothiazine-based ratiometric fluorescent probe was rationally designed and synthesized for the selective detection and imaging of sulfur dioxide derivatives (${\text{HSO}}_{3}^{－}$/${\text{SO}}_{3}^{2－}$). ${\text{HSO}}_{3}^{－}$/${\text{SO}}_{3}^{2－}$ induces aggregation-state modulation of probe diethyl (11-butyl-2-ethoxy-2-oxido-11H-[1,2]oxaphosphinino[6,5-b]phenothiazin-3-yl)phosphonate (T2), which causes a significant blue shift in its fluorescence emission maximum. Spectroscopic studies demonstrated that T2 exhibits high selectivity and sensitivity toward ${\text{HSO}}_{3}^{－}$/${\text{SO}}_{3}^{2－}$, with a detection limit as low as 3.96 μmol/L. Notably, T2 was successfully applied to the fluorescence imaging of exogenous ${\text{HSO}}_{3}^{－}$/${\text{SO}}_{3}^{2－}$ in living cells and plant tissues. In addition, T2-based test strips were developed for the rapid and visual detection of ${\text{HSO}}_{3}^{－}$/${\text{SO}}_{3}^{2－}$ in aqueous media. These results indicate that T2 represents a promising analytical tool for the detection and bioimaging of SO₂ derivatives in environmental and biological systems.

Key words: sulfur dioxide derivatives, phenothiazines, bioimaging, fluorescent probes

Cite this article

Zhongshan Xiao, Han Wang, Lu Xu, Ruijuan He, Guojing Yang, Yakun Wang, Zhaomin Liu, Tao Zhang. A Novel Phosphate-Ester-Phenothiazine Fluorescent Probe for Detection and Imaging of Sulfur Dioxide Derivatives[J]. Chinese Journal of Organic Chemistry, 2026, 46(8): 3068-3078.

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Fig. & Tab. 12

Scheme 1. Synthetic routes of probes T1 and T2

Scheme 2. Synthetic routes of probes T3 and T4

Figure 1. Photographs of probes T1 (a, b), T2 (c, d), T3 (e, f), and T4 (g, h) in the absence and presence of various analytes under daylight (left) and 365 nm UV irradiation (right) 1~22: probe, ${\text{PO}}_{4}^{3－}$, ${\text{HPO}}_{4}^{2－}$, ${\text{H}}_{2}{\text{PO}}_{4}^{－}$, ${\text{CO}}_{3}^{2－}$, ${\text{HCO}}_{3}^{－}$, ${\text{S}}_{2}{\text{O}}_{3}^{2－}$, ${\text{SO}}_{3}^{2－}$, ${\text{HSO}}_{3}^{－}$, ${\text{SO}}_{4}^{2－}$, F－, Cl－, Br－, I－, AcO－, ${\text{BH}}_{4}^{－}$, SCN－, ${\text{NO}}_{\text{2}}^{－}$, Zn2＋, Mg2＋, Al3＋, Ca2＋.

Figure 2. T2 (20 μmol/L) in pure aqueous solution: (a) UV absorption spectral changes, and (b) fluorescence spectral changes of T2 upon addition of 1.0 equiv. analyte; (c) UV-Vis spectral competition experiment of T2 with ${\text{HSO}}_{3}^{－}$/${\text{SO}}_{3}^{2－}$ at the ratio (A372/A425); (d) Fluorescence spectral competition experiment of T2 with ${\text{HSO}}_{3}^{－}$/${\text{SO}}_{3}^{2－}$ at the ratio (I515/I610) 1~22: probe, ${\text{PO}}_{4}^{3－}$, ${\text{HPO}}_{4}^{2－}$, ${\text{H}}_{2}{\text{PO}}_{4}^{－}$, ${\text{CO}}_{3}^{2－}$, ${\text{HCO}}_{3}^{－}$, ${\text{S}}_{2}{\text{O}}_{3}^{2－}$, ${\text{SO}}_{3}^{2－}$, ${\text{HSO}}_{3}^{－}$, ${\text{SO}}_{4}^{2－}$, F－, Cl－, Br－, I－, AcO－, ${\text{BH}}_{4}^{－}$, SCN－, ${\text{NO}}_{\text{2}}^{－}$, Zn2＋, Mg2＋, Al3＋, Ca2＋.

Figure 3. (a) Fluorescence spectral changes of T2 in aqueous solution upon treatment with increasing concentrations of ${\text{HSO}}_{3}^{－}$ (0~25 μmol/L); (b) Plot of the fluorescence intensity ratio (I515/I610) of probe T2 (20 μmol/L) versus ${\text{HSO}}_{3}^{－}$ concentration (0~22 μmol/L) in aqueous solution; (c) Linear relationship between I515/I610 and ${\text{HSO}}_{3}^{－}$ concentration in the range of 6~16 μmol/L

Figure 4. (a) Ultraviolet-visible absorption spectra of T2, T2- ${\text{HSO}}_{3}^{－}$, and the reference substance fluorescein in aqueous solution at room temperature (The dashed arrows indicate the excitation wavelength, 425 nm) used for the fluorescence assay; (b) Fluorescence emission spectra of each analyte under the same conditions at 425 nm excitation (Fluorescein was dissolved in 0.1 mol/L NaOH aqueous solution, while T2 and T2-${\text{HSO}}_{3}^{－}$ were dissolved in water. The spectra were labeled with their respective integral areas, AUC); (c) Fluorescence intensity time-dependent spectrum of probe T2 solution after addition of 1.0 equiv. ${\text{HSO}}_{3}^{－}$ (λex=425 nm)

Sample	Excitation wa- velength/nm	Absorbance (A_{425 nm})	Area under the curve	Φ_x/%
Fluorescein	425	0.045675	41565916	92.0%
T2	425	0.050134	6513340	13.1%
T2＋${\text{HSO}}_{3}^{－}$	425	0.047588	12128542	25.8%

Table 1. Relative fluorescence quantum yield of probe T2 and its bisulfite adduct

Figure 5. (a) 1H NMR spectrum of probe T2 in DMSO-d6; (b) 1H NMR spectrum of probe T2 after reaction with ${\text{HSO}}_{3}^{－}$ in DMSO-d6; (c) Fluorescence effect of T2 (10 μmol/L) in DMSO/H2O mixtures with different water fractions (fw) under a 365 nm UV lamp; (d) Fluorescence images of T2 (10 μmol/L) upon addition of ${\text{HSO}}_{3}^{－}$ (10 μmol/L) in DMSO/H2O mixtures with different water fractions (fw) under a 365 nm UV lamp; (e) TEM image of probe T2 in aqueous solution; (f) TEM image of the aqueous solution of probe T2 after reaction with ${\text{HSO}}_{3}^{－}$; (g) HRMS spectrum of probe T2 in water; (h) HRMS spectrum of probe T2 after reaction with ${\text{HSO}}_{3}^{－}$ in water

Figure 6. Survival rate of pancreatic cancer cell line PANC-1 after 48-hour culture in the presence of different concentrations of T2 (5, 10, 20 and 30 μmol/L)

Figure 7. Fluorescence images of PANC-1 cells incubated with T2 (20 μmol/L) at 37 ℃ followed by the addition of ${\text{HSO}}_{3}^{－}$ (40 μmol/L) Bright field (a, b, c), green channel (d, e, f), red channel (g, h, i), and merged images (j, k, l)

Figure 8. Sesame seedlings treated with probe T2 (20 μmol/L) and different concentrations of NaHSO3 solution: (a) Images under 365 nm UV lamp after 24-hour treatment; (b) Images under 365 nm UV lamp after 48-hour treatment

Figure 9. Photographs of probe T2 test strips after immersion in different analytes under (a) daylight and (b) 365 nm ultraviolet light

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