A ratiometric flavone-based fluorescent probe for hypochlorous acid detection with large Stokes shift and long-wavelength emission

Accepted Manuscript A ratiometric flavone-based fluorescent probe for hypochlorous acid detection with large Stokes shift and long-wavelength emission Long He, Yun Zhang, Haiqing Xiong, Jingpei Wang, Yani Geng, Benhua Wang, Yangang Wang, Zhaoguang Yang, Xiangzhi Song PII:

S0143-7208(19)30373-0

DOI:

https://doi.org/10.1016/j.dyepig.2019.03.029

Reference:

DYPI 7418

To appear in:

Dyes and Pigments

Received Date: 16 February 2019 Revised Date:

12 March 2019

Accepted Date: 13 March 2019

Please cite this article as: He L, Zhang Y, Xiong H, Wang J, Geng Y, Wang B, Wang Y, Yang Z, Song X, A ratiometric flavone-based fluorescent probe for hypochlorous acid detection with large Stokes shift and long-wavelength emission, Dyes and Pigments (2019), doi: https://doi.org/10.1016/j.dyepig.2019.03.029. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Graphical Abstract

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A flavone-based ratiometric fluorescent probe with large Stokes shift and long-wavelength emission for the sensitive and selective detection of hypochlorous acid has been developed. The application of this probe for imaging hypochlorous acid in zebra fish was successfully demonstrated under a single-wavelength excitation.

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A

ratiometric

flavone-based

fluorescent

probe

for

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hypochlorous acid detection with large Stokes shift and

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long-wavelength emission

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Long Hea‡, Yun Zhanga‡, Haiqing Xionga, Jingpei Wanga, Yani Geng a, Benhua Wanga*, Yangang

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Wangc*, Zhaoguang Yanga, b and Xiangzhi Songa,b*

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a

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Province, P. R. China, 410083.

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b

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Changsha, Hunan Province, P. R. China, 410083.

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College of Chemistry & Chemical Engineering, Central South University, Changsha, Hunan

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Key Laboratory of Hunan Province for Water Environment and Agriculture Product Safety,

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Province, China, 314001.

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Abstract

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A ratiometric fluorescent probe, FPT, was designed and synthesized for the selective

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and sensitive detection of hypochlorous acid (HClO) based on a flavone derivative

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under a single wavelength excitation. The addition of HClO to the solution of FPT

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induced a distinct fluorescence color change from orange to green. Probe FPT was

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successfully used to detect and image intracellular HClO in living zebra fish in a

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ratiometric manner.

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Keywords

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Ratiometric; Flavone-based; Long-wavelength; Large Stokes Shift

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College of Biological Chemical Science and Engineering, Jiaxing University, Jiaxing , Zhejiang

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1. Introduction

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Reactive oxygen species (ROS) are generated from molecular oxygen in different

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physiological pathways, and play vital roles in a wide range of biological and

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pathological processes[1-3]. As a typical ROS, hypochlorous acid (HClO) is known as

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a strong oxidant and displays antibacterial activity. Importantly, the biogenic HClO

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extensively participates in preventing inflammation and regulating cellular apoptosis

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to keep physiological homeostasis[4-7]. However, overexpressed HClO can consume

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antioxygen and give rise to the lopsidedness of intracellular oxidative stress[8], which

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results in cell damage and many diseases such as osteoarthritis, cardiovascular

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diseases, lung injury atherosclerosis, and cancer[9-13]. As a result, it is imperative to

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develop reliable and effective methods for monitoring and imaging HClO in living

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systems to elucidate its specific functions in pathophysiological process.

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Compared with other methods, fluorescent probes have been considered as a

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reliable tool for the real-time detection of HClO in living systems owing to its unique

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features such as high temporal-spatial and simplicity[14-23]. Besides the good

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sensitivity and selectivity, fluorescent probes with long-wavelength emissions are

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more suitable for biological imaging owing to the deep tissue penetration and low

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photo damage of long-wavelength photons[24-27]. Moreover, ratiometric fluorescent

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probes can self-calibrate through two dependent emissions and exhibit higher

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detection accuracy and better sensitivity than intensity-based ones[28-33].

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Flavone derivatives usually display a large Stokes shift owing to the excited state

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intramolecular proton transfer (ESIPT) process and have high fluorescent quantum 2

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developed to detect various species[34-37]. However, most of the flavone derivatives

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emit in a short-wavelength spectral region, which are subjected to the interference of

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auto-fluorescence from background, low tissue penetration and high photo damage to

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tissues[38-40].

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In this paper, we developed an orange-emitting flavone-based dye, FPT, with

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phenothiazine moiety as both the sensing group and the electron donor (Scheme

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1)[41]. In the presence of HClO, the electron-donating divalent sulfur atom of

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phenothiazine moiety is expected to be oxidized into an electron-withdrawing

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sulfoxide group[42], which would lead to a large spectral blue-shift due to the sharp

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change of ICT (intramolecular charge transfer) effect (Scheme 2). Meanwhile, the

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strong electron donating ability of phenothiazine group makes FPT have a

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long-wavelength emission. Therefore, FPT could serve as a fluorescent probe for

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HClO with a long-wavelength emission and a large Stokes shift in which

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phenothiazine moiety acted as a sensing group by virtue of the oxidation reaction.

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Scheme 1 Synthetic route of probe FPT. (a) 2-Hydroxyacetophenone, KOH, methanol, 65

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°C, 7 h, yield 22.5%. (b) 30% H2O2, 0.5 M NaOH, methanol, 70 °C, 3 h, yield 41.7%.

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Scheme 2 Proposed responding process of probe FPT toward HClO. 3

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2. Experiment section

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2.1. Materials and Instruments

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Reagents were purchased from commercial suppliers including Aladdin Industrial

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Corporation and Sinopharm Group Chemical Reagent Co., Ltd. 1H NMR and

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NMR spectra were recorded on a Bruker 400 NMR spectrometer with TMS as the

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internal standard. Absorption and emission spectra were recorded by a UV-2450

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spectrophotometer and an F-7000 fluorometer, respectively. Fluorescence imaging

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experiments were performed on an Olympus FV1000 confocal microscope. Zebra fish

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were obtained from Nanjing Eze-Rinka Ltd Co. China, and were cultured under an

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appropriate condition.

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2.2. Preparation of Probe and Analytes

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The stock solution of the probe in CH3CN was prepared at a concentration of 1.0 ×

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10-3 M. The analytes of ROS [(hypochlorite (NaClO), singlet oxygen (1O2), hydrogen

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peroxide (H2O2), superoxide (O2-), nitric oxide (NO), hydroxyl radical (.OH),

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peroxynitrite (ONOO-), organic peroxide radicals (ROO.), butylhydroperoxide

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(TBHP)] and other ions were prepared according to the reported methods[43-45]. The

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test solutions of probe FPT (10.0 µM) were prepared by mixing appropriate amounts

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of the stock solution of probe FPT, 20 mM HEPES buffer (CTAB 1.0 mM, pH = 7.4)

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and respective analytes.

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2.3. Cell Cytotoxicity Assay

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eagle’s medium (DMEM) for 24 h at an appropriate density. MTT assays was

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performed by incubation cells with the solution of probe FPT at different

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concentrations (0, 5, 10, 15, and 20 µM) for 24 h.

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2.4. Synthesis of Compound PTZ

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Compound PTZ was obtained according to the literature method[46].

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2.5. Synthesis of Compound 1

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Potassium hydroxide (25.0 mg, 0.45 mmol) was added to a solution of PTZ (56.0 mg,

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0.2 mmol) and 2-hydroxyacetophenone (47.0 mg, 0.35 mmol) in 5 mL dry methanol.

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Then the reaction mixture was refluxed for 7 h. After removing the solvent under a

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reduced pressure, the crude product was purified by column chromatography using

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petroleum ether/dichloromethane (8/1, v/v) as eluent to afford compound 1 as a dark

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red solid (18.0 mg, 22.5%). HRMS (ESI) m/z: calcd for C25H22NO2S [M-H]-,

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400.1377; found, 400.1387. 1H NMR (500 MHz, CDCl3) δH 12.92 (s, 1H), 7.92 (d, J =

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7.7 Hz, 1H), 7.85 (d, J = 14.3 Hz, 1H), 7.52 – 7.43 (m, 3H), 7.38 (d, J = 5.9 Hz, 1H),

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7.13 (d, J = 7.2 Hz, 2H), 7.02 (d, J = 8.3 Hz, 1H), 6.94 (t, J = 7.5 Hz, 1H), 6.84 (d, J =

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7.1 Hz, 3H), 4.04 (s, 2H), 1.85 – 1.73 (m, 2H), 1.48 (dd, J = 14.8, 7.4 Hz, 2H), 0.97 (t,

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C NMR (100 MHz, CDCl3) δC 163.5, 147.8, 144.5, 143.9, 136.2,

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J = 7.3 Hz, 3H).

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129.5, 129.3, 128.8, 127.5, 126.7, 125.1, 123.7, 123.1, 120.1, 118.8, 118.6, 117.5,

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115.7, 115.2, 47.4, 28.8, 20.1, 13.8.

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2.6. Synthesis of Probe FPT 5

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methanol and 3 mL 0.5 M sodium hydroxide aqueous solution was slowly added 100

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µL 30% H2O2. The resulting mixture was heated at 70 °C for 3 h and then poured into

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20 mL cold water. Next, 1 M HCl was added into the obtained mixture to adjust pH to

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2-3. Finally, the mixture was extracted with dichloromethane (20.0 mL × 3), and the

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organic layers were collected and dried over anhydrous sodium sulfate. Removed the

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organic solvent in vacuum to give the crude product, which was further purified by

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column chromatography (petroleum ether/dichloromethane, v/v, 6/1) to yield probe

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FPT (50.0 mg, 41.7%) as an orange solid. HRMS (ESI) m/z: calcd for C25H21NO3S

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[M+Na]+, 438.1134; found, 438.1123. 1H NMR (400 MHz, CDCl3) δH 8.23 (d, J = 7.8

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Hz, 1H), 8.11 (d, J = 8.2 Hz, 1H), 8.00 (s, 1H), 7.68 (t, J = 7.7 Hz, 1H), 7.57 (d, J =

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8.4 Hz, 1H), 7.40 (t, J = 7.4 Hz, 1H), 7.14 (d, J = 6.9 Hz, 2H), 7.03 – 6.92 (m, 3H),

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6.89 (d, J = 7.8 Hz, 1H), 3.91 (s, 2H), 1.83 (dt, J = 14.4, 7.2 Hz, 2H), 1.49 (dd, J =

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14.9, 7.4 Hz, 2H), 0.97 (t, J = 7.3 Hz, 3H).

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144.5, 137.7, 133.5, 127.2, 125.9, 125.6, 124.5, 123.7, 122.8, 121.0, 118.0, 115.6,

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114.7, 47.3, 29.7, 28.8, 20.1, 13.8.

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3. Results and discussion

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3.1. Optical Properties of Probe FPT

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The optical response of probe FPT toward HClO was investigated in 20 mM HEPES

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buffer solution (1.0 mM CTAB, pH = 7.4) at 25 ℃. The solution of free probe FPT

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displayed an intense absorbance peak at 446 nm and a main emission band at 586 nm

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C NMR (100 MHz, CDCl3) δC 155.2,

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ACCEPTED MANUSCRIPT (Figs. 1 and S1). And the Stokes shift is 140 nm. The treatment of probe FPT with

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HClO triggered a prominent color change from yellow to colorless, which was

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sensitive to naked eyes (Fig. S1); and the fluorescence changed from orange (λmaxEm =

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586 nm) to green (λmaxEm = 524 nm). Upon the gradual addition of HClO from 0 to 8.0

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equiv. to the solution of probe FPT, the emission peak at 586 nm notably decreased

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and a new emission band at 524 nm emerged (Fig. 1).

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Fig. 1 Fluorescence spectra of probe FPT (10.0 µM) toward HClO in 20 mM HEPES buffer

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solution (1.0 mM CTAB, pH = 7.4) at 25 °C. Excitation wavelength: 380 nm. Inset: fluorescence

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photos of probe FPT (10.0 µM) before and after the treatment with HClO under a 365 nm lamp.

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The ratio of the fluorescence intensities at 524 nm and 586 nm (I524/I586) showed

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a good linear relationship with the concentration of HClO in a range of 0 to 6.0 equiv.

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(Fig. 2). The detection limit of probe FPT towards HClO was calculated to be 6.6 nM

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based on the signal to noise (S/N = 3), indicating the high sensitivity of probe FPT.

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Additionally, the time-dependent fluorescence experiment on probe FPT with HClO

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revealed that the intensity ratio (I524/I586) maximized in a short time (less than 10 min,

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Fig. 3). From the above results, we could conclude that probe FPT was able to

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ACCEPTED MANUSCRIPT qualitatively and quantitatively monitor HClO in aqueous media with a ratiometric

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model. It’s noteworthy that the ratiometric detection was performed under a

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single-wavelength excitation, which could simplify the sensing process and improve

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the detection sensitivity and accuracy.

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Fig. 2 The linear relationship between the intensity ratio (I524/I586) of probe FPT (10.0 µM) and

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the concentration of HClO (0.0-60.0 µM) in 20 mM HEPES buffer solution (1.0 mM CTAB, pH =

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7.4) at 25 °C. Excitation wavelength: 380 nm.

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Fig. 3 The intensity ratio (I524/I586) of probe FPT (10.0 µM) in the absence/presence of HClO (8.0

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equiv.) in 20 mM HEPES buffer solution (1.0 mM CTAB, pH = 7.4) as a function of time at 25 °C.

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Excitation wavelength: 380 nm.

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Next, we explored whether the probe FPT could selectively detect HClO over the

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relevant analytes including ROS/RNS [hypochlorite (NaClO), singlet oxygen (1O2),

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hydrogen peroxide (H2O2), superoxide (O2-), nitric oxide (NO), hydroxyl radical

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(.OH), peroxynitrite (ONOO-), organic peroxide radicals (ROO.), butylhydroperoxide

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(TBHP)], and other analytes (Hcy, Cys, GSH, SH-, HSO3-, S2O32-, SO42-, Br-, I-, K+,

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Na+, Fe3+). As illustrated in Figs. 4 and 5, negligible fluorescence change was seen in

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the presence of the relevant species.

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Fig. 4 Fluorescence spectra of probe FPT (10.0 µM) in response to relevant testing species (8.0

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equiv. for each) in 20 mM HEPES buffer solution (1.0 mM CTAB, pH = 7.4) with an excitation

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wavelength at 380 nm at 25 °C. Test species: (1) PBS, (2) ClO-, (3)1O2, (4) H2O2, (5) O2-, (6) NO.,

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(7) .OH, (8) ONOO-, (9) ROO., (10) TBHP, (11) Hcy, (12) Cys, (13) GSH, (14) SH-, (15) HSO3-,

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(16) S2O32-, (17) SO42-, (18) Br-, (19) I-, (20) K+, (21)Na+, (22) Fe3+.

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Fig. 5 The ratio of fluorescence intensities of probe FPT (10.0 µM) (I525/I586) in the presence of

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different species (8.0 equiv.) in 20 mM HEPES buffer solution (1.0 mM CTAB, pH = 7.4) at 25 °C.

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(1) PBS, (2) ClO-, (3)1O2, (4) H2O2, (5) O2-, (6) NO., (7) .OH, (8) ONOO-, (9) ROO., (10) TBHP,

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(11) Hcy, (12) Cys, (13) GSH, (14) SH-, (15) HSO3-, (16) S2O32-, (17) SO42-, (18) Br-, (19) I-, (20)

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K+, (21)Na+, (22) Fe3+. Excitation wavelength: 380 nm.

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3.3. Mechanism

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As described above, the sensing of HClO by probe FPT was realized by the oxidation

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of bivalent sulphur atom in phenothiazine moiety. In order to verify the sensing

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mechanism, mass spectral analysis was carried out on the mixture of probe FPT and

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HClO. A peak at 430.1118 was found in the mass spectrum, which was equal to the

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accurate molecular weight of compound FPT-O. This result strongly supported the

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responding mechanism proposed in Scheme 2.

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3.4 The Effect of pH

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To confirm whether probe FPT can be applied in physiological system, pH effect on

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the performance of this probe toward HClO was investigated from pH 2 to 13. As

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shown in Fig. 6, the intensity ratio (I524/I586) of probe FPT remained unchanged in a

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ACCEPTED MANUSCRIPT wide pH range in the absence of HClO, but drastically increased in a pH range from

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6.0 to 10.0 in the presence of HClO. These results indicated that probe FPT had a

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good stability and could function well for detecting HClO under physiological

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condition.

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Fig. 6 The intensity ratio (I524/I586) of probe FPT (10.0 µM) at different pH values in the

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absence/presence of HClO at 25 °C. Excitation wavelength: 380 nm.

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3.5 The Bioimaging Applications

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The excellent properties of FPT in aqueous solutions suggested it hold a potential to

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detect HClO in biological systems. Thus, MTT assays were performed on living HeLa

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cells with probe FPT, and it was found that cells had high survival rates when treated

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with probe FPT at a concentration below 20.0 µM (Fig S3). Inspired by the low

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toxicity of probe FPT, imaging HClO by probe FPT was carried out in zebra fish.

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When zebra fish was incubated with probe FPT (10.0 µM), strong red fluorescent

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signals were observed from channel 1 (570-650 nm) and weak green fluorescence was

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found from channel 2 (420-540 nm), indicating a low-level of HClO in zebra fish (Fig.

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7A). In contrast, when zebra fish was pretreated with HClO (8.0 equiv.) for 30 min

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fluorescence was displayed from channel 1 and bright green fluorescence was

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observed from channel 2 (Fig. 7B). These results implied that probe FPT was a

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potential tool for detecting HClO in living organisms.

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Fig. 7 Fluorescence and bright filed images of zebra fish. A1-A4: Zebra fish incubated with probe

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FPT (10.0 µM) for 30 min at 25 °C. B1-B4: Zebra fish pre-treated with HClO (8.0 equiv.) for 30

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min at 25 °C, then incubated with probe FPT (10.0 µM) for another 10 min at 25 °C. (Ch1:

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570-650 nm; Ch2: 420-540 nm; Ex = 405 nm).

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4. Conclusions

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In summary, a ratiometric fluorescent probe FPT for HClO was developed based on a

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flavone dye. This probe displayed good selectivity, excellent sensitivity,

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long-wavelength emissions as well as a large Stokes shift. The oxidation of

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phenothiazine moiety in this probe by HClO remarkably adjusted the intramolecular

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charge transfer process, which induced a sharp fluorescence change, and thereby

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realized a ratiometric detection. Importantly, probe FPT had been successfully used

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for detecting intracellular HClO in living organisms.

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5. Acknowledgements

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ACCEPTED MANUSCRIPT This work was supported by the National Natural Science Foundation of China (No.

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U1608222), Special Fund for Agro-scientific Research in the Public Interest of China

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(No. 201503108) and the State Key Laboratory of Chemo/Biosensing and

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Chemometrics (2016005), the Fundamental Research Funds for the Central

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Universities of Central South University (2018zzts364).

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Research Highlights This probe had large Stokes shift.

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This probe displayed a ratio fluorescence I524 nm/I586 nm with a single-wavelength excitation at 380 nm.

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This probe had long-wavelength emission.

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