A New Fluorescent Probe for Hypochlorous Acid Based on Chlorinium Ions Recognition Mechanism and Its Bioimaging Research in Living Cells

doi:10.6023/cjoc202101038

Abstract

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

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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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Fig. & Tab. 9

Scheme 1. Synthesis and Proposed mechanisms of probe PIOCl

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)

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

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

Figure 4. HRMS spectrum of PIOCl with HOCl

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 +

Figure 6. 1H NMR spectrum of PIOCl with HOCl

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

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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