基于亲电氯代反应的次氯酸荧光探针构建及细胞成像研究

doi:10.6023/cjoc202101038

摘要/Abstract

设计并合成了一种简单的由吩噻嗪-茚酮共轭的新型次氯酸荧光探针PIOCl. 该探针实现了在生理条件下, 基于氯正离子(Cl⁺)引发独特亲电氯代反应机制的HOCl检测. 实验结果表明, 探针PIOCl对HOCl具有荧光“关-开”型响应, 检测前后荧光强度增加了600多倍, HOCl浓度在0~6.0 μmol/L呈良好的线性关系(R²=0.997), 检测限达1.40 nmol/L. 可实现低至0.25 μmol/L的裸眼检测以及快速响应(<3 s). 通过质谱实验、光谱实验和理论计算验证了 PIOCl对HOCl的识别机理. 利用荧光成像技术, 成功对细胞内源性和外源性HOCl进行了检测, 表明探针PIOCl在生物学上具有潜在的应用前景.

关键词: 次氯酸, 荧光探针, 亲电氯代, 茚酮, 荧光增强

A simple phenothiazine-indanone conjugated fluorescent probe PIOCl for HOCl was designed and synthesized. For the first time, a new fluorescent probe for HOCl based on Cl⁺ induced the unique electrophilic substitution reaction under the physiological conditions. The probe PIOCl was a turn-on response to HOCl, and the fluorescence intensity increased more than 600 times after addition of HOCl. In the range of HOCl concentration from 0 to 6.0 μmol/L, the fluorescence intensity had a good linear relationship with HOCl concentration ( R²=0.997), the detection limit was 1.40 nmol/L, and the naked eye detection as low as 0.25 μmol/L and rapid response (<3 s) can be achieved under 1.0 μmol/L HOCl. The sensing mechanism of probe PIOCl for HOCl was verified by NMR, HRMS and density function theory calculations. Using fluorescence imaging technology, the endogenous and exogenous HOCl has been successfully detected, which indicates that probe PIOCl has potential application prospects in analytical detection and pathological analysis.

Key words: hypochlorous acid, fluorescent probe, electrophilic chlorination, indanone, turn-on mechanism

引用此文

赵云, 李艳芳, 李蓉晓, 王雅卿, 樊晓霞. 基于亲电氯代反应的次氯酸荧光探针构建及细胞成像研究[J]. 有机化学, 2021, 41(5): 1974-1981.

Yun Zhao, Yanfang Li, Rongxiao Li, Yaqing Wang, Xiaoxia Fan. A New Fluorescent Probe for Hypochlorous Acid Based on Chlorinium Ions Recognition Mechanism and Its Bioimaging Research in Living Cells[J]. Chinese Journal of Organic Chemistry, 2021, 41(5): 1974-1981.

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图/表 9

图式1. 荧光探针PIOCl的合成路线及机理

Scheme 1. Synthesis and Proposed mechanisms of probe PIOCl

图1. 在PBS缓冲溶液(20 mmol/L, pH=7.4, 30% EtOH)中探针PIOCl (10 μmol/L)与不同浓度的HOCl (0~10 μmol/L)的(a)紫外-可见光吸收光谱和(b)荧光发射光谱, (c)探针PIOCl (10 μmol/L)与低浓度HOCl (0~1.0 μmol/L)的荧光发射光谱[插图: 探针 PIOCl (10 μmol/L)分别与0.0、0.25、0.5、1.0和10.0 μmol/L HOCl)在365 nm紫外灯照射下的照片], 以及(d)探针PIOCl在514 nm处荧光强度与HOCl浓度的线性相关性(λ ex=420 nm)

Figure 1. In PBS buffer (pH=7.4, 10 mmol/L, 30% EtOH), absorption spectra (a) and fluorescence spectra (b) of probe PIOCl (10 μmol/L) in response to HOCl(0~10 μmol/L), (c) fluorescence spectra at low concentration of HOCl (0~1.0 μmol/L) (insert: the color changes after adding HOCl under 365 nm UV-light), and (d) the linear relationship between the fluorescence intensity at 514 nm and the concentration of HOCl (0~6 μmol/L)

图2. (a)荧光强度随时间变化曲线(λex=420 nm, 探针PIOCl 10 μmol/L, HOCl分别为0, 1.0, 2.0, 5.0和10 μmol/L), 以及(b)探针PIOCl (10 μmol/L)及探针PIOCl (10 μmol/L)与HOCl (10 μmol/L)荧光响应受pH的影响

Figure 2. (a) Time-dependent fluorescent intensities of probe PIOCl treated with HOCl (λex=420 nm, PIOCl 10 μmol/L, HOCl 0, 1.0, 2.0, 5.0, and 10 μmol/L), and (b) plot of fluorescence intensity of probe PIOCl (10.0 μmol/L) in the absence/ presence of HOCl (10 μmol/L) at different pH

图3. (a)探针PIOCl与各种分析物相互作用后的荧光光谱图, 以及(b)在514 nm处各种分析物的荧光响应强度

Figure 3. (a) Fluorescence spectra of probe PIOCl in the presence of different biological relevant analytes and (b) fluorescent intensity changes of probe PIOCl in the presence of different biological relevant analytes at 514 nm The numbers represent: (1) probe PIOCl (1.0×10–5 mol/L), (2) MgCl2, (3) Zn(NO3)2, (4) Ca(NO3)2, (5) Cu(NO3)2, (6) Fe(NO3)3, (7) NaF, (8) NaNO3, (9) NaNO2, (10) Na2S, (11) Cys, (12) GSH, (13) H2O2, (14) ?OH, (15) ?$\mathrm{O}_{2}^{-}$, (16) 1O2, (17) t-BuOO–, (18) NO?, (19) ONOO-, (20) HOBr, (21) NaClO. λex=420 nm

图4. 探针PIOCl与HOCl作用后的高分辨质谱

Figure 4. HRMS spectrum of PIOCl with HOCl

图5. 探针PIOCl与HOCl作用后受酸影响的紫外可见吸收光谱图

Figure 5. UV-Vis absorption spectra of probe PIOCl (10.0 μmol/L) to HOCl (10.0 μmol/L) in the absence and presence of H +

图6. 探针PIOCl与HOCl反应后1H NMR谱

Figure 6. 1H NMR spectrum of PIOCl with HOCl

图7. 探针PIOCl和PIOCC的优化分子结构和前线轨道能量

Figure 7. The Optimized structures and frontier orbital energy of PIOCl and PIOCC

图8. 探针PIOCl在HeLa活细胞中的荧光成像

Figure 8. Live cell fluorescence imaging of probe PIOCl in living HeLa cells (a~b) Living HeLa cells incubation with PIOCl (10 μmol/ L) for 30 min, and (c~d) living HeLa cells incubation with PIOCl (10 μmol/L) and HOCl (10 μmol/L) for 30 min

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