用于二氧化硫衍生物检测与成像的新型磷酸酯-吩噻嗪荧光探针

doi:10.6023/cjoc202603043

摘要/Abstract

设计并合成了一种基于吩噻嗪荧光团的新型比率型荧光探针(11-丁基-2-乙氧基-2-氧代-11H-[1,2]氧杂膦杂苯并[6,5-b]吩噻嗪-3-基)膦酸二乙酯(T2), 用于SO₂衍生物(${\text{HSO}}_{3}^{－}$/${\text{SO}}_{3}^{2－}$)检测和成像. ${\text{HSO}}_{3}^{－}$/${\text{SO}}_{3}^{2－}$可以诱导探针T2自组装聚集, 引起探针T2的最大发射峰蓝移, 从而实现对SO₂衍生物的比率型检测. 实验结果表明, T2对${\text{HSO}}_{3}^{－}$/${\text{SO}}_{3}^{2－}$具有优异的选择性, 检测限低至3.96 μmol/L. 该探针不仅能够实现活细胞以及植物中外源性二氧化硫衍生物的成像, 基于T2制备的检测试纸, 可实现水溶液中的${\text{HSO}}_{3}^{－}$/${\text{SO}}_{3}^{2－}$的快速可视化检测. 综上, 探针T2在SO₂衍生物检测及生物成像领域可成为一种有力工具.

关键词: 二氧化硫衍生物, 吩噻嗪, 生物成像, 荧光探针

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

引用此文

肖忠闪, 王涵, 徐露, 何瑞娟, 杨国静, 王亚坤, 刘兆敏, 张涛. 用于二氧化硫衍生物检测与成像的新型磷酸酯-吩噻嗪荧光探针[J]. 有机化学, 2026, 46(8): 3068-3078.

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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图/表 12

图式1. 探针分子T1和T2的合成路线

Scheme 1. Synthetic routes of probes T1 and T2

图式2. 探针分子T3和T4的合成路线

Scheme 2. Synthetic routes of probes T3 and T4

图1. 探针T1 (a, b)、探针T2 (c, d)、探针T3 (e, f)和探针T4 (g, h)中加入不同分析物的日光(左)和365 nm紫外光下(右)照片

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＋.

图2. T2 (20 μmol/L)在纯水溶液中: 加入1.0 equiv.分析物后T2的(a)紫外吸收光谱变化和(b)荧光光谱变化; (c)在比值(A372/A425)下T2与${\text{HSO}}_{3}^{－}$/${\text{SO}}_{3}^{2－}$的紫外-可见光谱竞争实验; (d)在比值(I515/I610)下T2与${\text{HSO}}_{3}^{－}$/${\text{SO}}_{3}^{2－}$的荧光光谱竞争实验

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＋.

图3. (a) 0~25 μmol/L ${\text{HSO}}_{3}^{－}$处理后T2在水溶液中的荧光光谱变化; (b)在水溶液中探针T2 (20 μmol/L)与添加${\text{HSO}}_{3}^{－}$ (0~22 μmol/L)后的荧光强度比(I515/I610); (c)荧光强度比(I515/I610)与${\text{HSO}}_{3}^{－}$ (6~16 μmol/L)浓度的线性关系

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

图4. (a) T2、T2-${\text{HSO}}_{3}^{－}$及参比物质荧光素(Fluorescein)在室温水溶液中的紫外-可见吸收光谱(虚线箭头标注为用于荧光测试的激发波长, 425 nm); (b)相同条件下各待测物在425 nm激发下的荧光发射光谱(荧光素溶于0.1 mol/L NaOH水溶液, T2和T2-${\text{HSO}}_{3}^{－}$溶于水, 图中标注为各光谱的积分面积, AUC); (c)加入1.0 equiv. ${\text{HSO}}_{3}^{－}$后探针T2溶液的荧光强度随时间变化光谱图(λex=425 nm)

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%

表1. 探针T2及其亚硫酸氢盐加合物的相对荧光量子产率

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

图5. (a)探针T2的1H NMR (DMSO-d6); (b)探针T2响应${\text{HSO}}_{3}^{－}$后的1H NMR (DMSO-d6); (c) 365 nm紫外灯下T2 (10 μmol/L)在不同水分数(fw)的DMSO/H2O混合物中的荧光图; (d) 365 nm紫外灯下T2 (10 μmol/L)在添加${\text{HSO}}_{3}^{－}$(10 μmol/L)后在不同水分数(fw)的DMSO/H2O混合物中的荧光效应; (e)探针T2在水溶液中的TEM图; (f)探针T2与${\text{HSO}}_{3}^{－}$响应后水溶液的TEM图; (g)水相中探针T2的HRMS; (h)水相中探针T2响应${\text{HSO}}_{3}^{－}$后的HRMS

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

图6. 胰腺癌细胞系PANC-1细胞在不同浓度的T2 (5, 10, 20和30 μmol/L)存在下培养48 h后的存活率

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)

图7. PANC-1细胞在37 ℃下加入T2 (20 μmol/L)再加入${\text{HSO}}_{3}^{－}$ (40 μmol/L)孵育后的荧光图

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)

图8. 用探针T2 (20 μmol/L)和不同浓度的NaHSO3溶液处理的芝麻幼苗: (a)处理24 h后365 nm紫外灯下的图像; (b)处理48 h后365 nm紫外灯下的图像

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

图9. 探针T2试纸条在浸入不同分析物后在(a)日光下(b) 365 nm紫外光下的拍摄照片

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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