Construction of a ratiometric fluorescent probe with an extremely large emission shift for imaging hypochlorite in living cells

Accepted Manuscript Construction of a ratiometric fluorescent probe with an extremely large emission shift for imaging hypochlorite in living cells

Xuezhen Song, Baoli Dong, Xiuqi Kong, Chao Wang, Nan Zhang, Weiying Lin PII: DOI: Reference:

S1386-1425(17)30569-3 doi: 10.1016/j.saa.2017.07.011 SAA 15298

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received date: Revised date: Accepted date:

7 April 2017 5 July 2017 11 July 2017

Please cite this article as: Xuezhen Song, Baoli Dong, Xiuqi Kong, Chao Wang, Nan Zhang, Weiying Lin , Construction of a ratiometric fluorescent probe with an extremely large emission shift for imaging hypochlorite in living cells, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2017), doi: 10.1016/j.saa.2017.07.011

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ACCEPTED MANUSCRIPT Construction of a ratiometric fluorescent probe with an extremely large emission shift for imaging hypochlorite in living cells Xuezhen Song, Baoli Dong, Xiuqi Kong, Chao Wang, Nan Zhang and Weiying Lin*

Institute of Fluorescent Probes for Biological Imaging, School of Chemistry and Chemical Engineering, School of

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Materials Science and Engineering, University of Jinan, Shandong 250022, P.R. China.

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E-mail: [email protected]

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Abstract:

Hypochlorite is one of the important reactive oxygen species (ROS) and plays critical roles in

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many biologically vital processes. Herein, we present a unique ratiometric fluorescent probe (CBP) with an extremely large emission shift for detecting hypochlorite in living cells. Utilizing

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positively charged α,β-unsaturated carbonyl group as the reaction site, the probe CBP itself

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exhibited near-infrared (NIR) fluorescence at 662 nm, and can display strong blue fluorescence at 456 nm when responded to hypochlorite. Notably, the extremely large emission shift of 206 nm

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could enable the precise measurement of the fluorescence peak intensities and ratios. CBP showed

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high sensitivity, excellent selectivity, desirable performance at physiological pH, and low cytotoxicity. The bioimaging experiments demonstrate the biological application of CBP for the ratiometric imaging of hypochlorite in living cells.

Key words: Fluorescent probe; Hypochlorite; Ratiometric fluorescence imaging

1

ACCEPTED MANUSCRIPT 1. Introduction Hypochlorite (ClO-), as one of the important reactive oxygen species (ROS) in living biological system, is genarally produced from the peroxidation of chloride ions catalyzed by enzyme myeloperoxidase (MPO) mainly in leukocytes including

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monocytes, macro-phages and neutrophils [1-5]. Hypochlorite plays critical roles in

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many biologically vital processes especially in the immune system, and may also

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induce tissue injury due to the destruction of proteins, nucleic acids, and lipids by the oxidation and chlorination reactions [6-8]. On the other hand, the excessive

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production of hypochlorite owing to upregulation in MPO levels can lead to a variety

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of diseases, such as cardiovascular diseases, neuron degeneration, arthritis, and cancer [9-12]. Therefore, it is of great interest to detect hypochlorite levels for exploring its

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fluctuations in living organisms.

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Fluorescence imaging is an attractive method for sensing biomolecules in living system due to its high sensitivity, excellent selectivity and real-time analysis [13-16].

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Until now, many fluorescent probes for sensing hypochlorite with excellent properties

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have been developed [17-22]. However, most of them are intensity-based probes which tend to be disturbed by the variations in excitation intensity, inhomogeneous cellular distribution, probe concentration, etc. By contrast, ratiometric fluorescent probes allow the measurement of the ratio of the emission intensities at two different wavelengths, which could provide a built-in correction for environmental effects and be benefit for the quantitative measurements [23]. However, to our best knowledge, a few ratiometric fluorescentprobes for hypochlorite with relative small emission shifts 2

ACCEPTED MANUSCRIPT (generally less than 130 nm) were reported. A large emission shift for ratiometric fluorescent probe could decrease the fluorescence detection errors resulted from the spectral overlap of the probe and the product after responding to analyte, and is favorable for the precise measurement of the peak intensities and ratios [24-28].

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emission shift for sensing hypochlorite in living system.

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Therefore, there is a great demand to develop ratiometric fluorescent probe with large

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Herein, we present a unique ratiometric fluorescent probe with extremely large emission shift for imaging hypochlorite in living cells. The probe itself exhibited

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near-infrared fluorescence at 662 nm, and can display strong blue fluorescence at 456

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nm in response to hypochlorite. Notably, the extremely large emission shift of 206 nm could enable the precise measurement of the intensities and ratios of the fluorescence

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peaks. The probe showed high sensitivity, excellent selectivity, desirable performance

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at physiological pH and low cytotoxicity. The bioimaging experiments demonstrate that this probe can be successfully applied for the ratiometric imaging of hypochlorite

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in living cells.

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2. Experimental

2.1. Materials and instruments. Unless otherwise stated, all solvents and reagents were commercially available and used without further purification unless for special needs. Twice-distilled water was used in all the experiments. Melting point was determined on a Kofler hot-stage microscope melting point apparatus and was uncorrected. The fluorescence spectra and relative fluorescence intensity were measured with a Hitachi F-4600 3

ACCEPTED MANUSCRIPT spectrofluorimeter with a 10 mm quartz cuvette. UV/vis spectra were made with a Shimadzu UV-2700 spectrophotometer. High-resolution mass spectra (HRMS) were collected using a Bruker apex-Ultra mass spectrometer (Bruker Daltonics Corp., USA) in electrospray ionization (ESI) mode. Mass spectra (MS) were collected using 13

C NMR

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Agilent 6510 Q-TOF LC/MS for studying response mechanism. 1H and

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spectra were recorded on Bruker AVANCE III 400 MHz Digital NMR Spectrometer,

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using tetramethylsilane (TMS) as internal reference. Cells imaging was performed with a Nikon A1R confocal microscope. TLC analysis was performed on silica gel

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plates and column chromatography was conducted over silica gel (mesh 200-300),

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both of which were obtained from the Qingdao Ocean Chemicals.

2.2. DFT calculations.

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The ground state structure of CS-A was optimized using DFT with B3LYP functional and 6-31G basis set. The initial geometries of the compounds were generated by the

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GaussView software. The excited state related calculations (UV-vis absorption) were carried out with the time-dependent DFT (TD-DFT) with the optimized structure of

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the ground state (DFT/6-31G). The emission of the fluorophores was calculated based on the optimized S1 excited state geometry. All of these calculations were performed with Gaussian 09 software.

2.3. Synthesis of the probe CBP. 4-Diethylamino-2-hydroxy-benzaldehyde 3-acetyl-7-hydroxy-chromen-2-one

(193

(204

mg, 4

1

mg,

1

mmol)

were

mmol) dissolved

and in

ACCEPTED MANUSCRIPT methanesulfonic acid (3 mL) and stirred at 90 ℃for 8 h. After being cooled to room temperature, ice (10 g) and 70% perchloric acid (1.0 mL) was added. The resulting solution was filtered, and washed with water to afford the crude product. The crude product was purified by silica gel flash chromatography by using CH2Cl2/CH3OH

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(15:1) as eluent to afford dark blue product CBP perchlorate (382 mg, 83.0 %). mp:

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267-269 ℃. 1H NMR (DMSO-d6, 400 MHz): δ 9.23 (s, 1 H), 8.72 (d, 1 H, J = 8.0 Hz),

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8.30 (d,1 H, J = 8.0 Hz), 8.01 (d, 1 H, J = 12.0 Hz), 7.86 (d, 1 H, J = 8.0 Hz), 7.52 (dd, 1 H, J1 = 4.0 Hz, J2 = 8.0 Hz), 7.31 (d, 1 H, J = 4.0 Hz), 6.97 (dd, 1 H, J1 = 4.0 Hz, J2

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= 8.0 Hz ), 6.85 (d, 1 H, J = 4.0 Hz), 3.72 (q, 4H, J = 8.0 Hz), 1.30 (t, 6H, J = 8.0 Hz). 13

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C NMR (DMSO-d6, 100 MHz): δ 166.16, 161.10, 159.53, 157.85, 157.35, 156.95,

148.99, 147.46, 133.28, 133.15, 120.05, 119.30, 115.72. 112.08, 111.92, 111.39,

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102.59, 96.12, 46.34, 12.61. HRMS (ESI): calcd for [M]+ 362.1387, found 362.1382.

2.4. Spectroscopic studies of the probe CBP.

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DEA/NONOate (diethylamine/NONOate), tert-butyl hydroperoxide (TBHP) were obtained from commercial sources and used without additional purification.

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Twice-distilled water and spectroscopic grade EtOH were used for spectroscopic studies. Nitric oxide (NO) was generated from DEA/NONOate (stock solution 1 mM in 0.01M NaOH). The nitric oxide (NO) stock solution in de-ionized water was prepared by bubbling NO into deoxygenated de-ionized water for 15 min. The other analytes were added to the solution of probe (final concentration, 5 μM) in PBS (20 mM, pH 7.4, 5% EtOH). The resulting solution was kept at room temperature, and then the fluorescence intensities were recorded with excitation at 410 nm unless for 5

ACCEPTED MANUSCRIPT special needs.

2.5. Cell toxicity assay. The MTT assay was used to evaluate the cytotoxicity of probe. HeLa cells were seeded onto 96-well plates at a density of 1×104 cells/well and incubated for 24 h. The

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medium was replaced by various probe over a range of concentrations (1~20 μM)

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dissolved in culture medium. After a 4h incubation, 10 μL MTT (5 mg/mL in PBS)

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were added and the cells were incubated for 4 h. After that, the medium was removed,

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and 100μL of DMSO were added to dissolveformazan crystals. The plate was agitated for 10 min, and each well was finally analyzed by the microplate reader (Thermo

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Scientific, Multiskan MK3) and detected by the absorbance at 570 nm.

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2.6. Cell culture and fluorescence imaging.

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HeLa cells were cultured in minimum essential medium (MEM) supplemented with 10% FBS (fetal bovine serum) and incubated at 37 °C in 5% CO2/air atmosphere. For

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fluorescence imaging, Hela cells were seeded into culture dishes with appropriate density and cultured in culture medium. After 24 h, the cells were first incubated with

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10 μM CBP for 30 min at 37 °C and washed three times with PBS (20 mM, pH 7.4) to remove excess extracellular dye. Then the cells were treated with NaClO (100 μM) at 37 °C for another 30 min. Fluorescence images were acquired with Nikon A1R confocal microscope with an objective lens (20 ×). The excitation wavelengths were set to 405 nm and 647 nm for blue and red channels, respectively.

3. Results and Discussion 6

ACCEPTED MANUSCRIPT 3.1. Design ans synthesis of the probe CBP. To rationally design a new ratiometric fluorescent probe with large emission shift for sensing hypochlorite, a selective chemical reaction mediated by hypochlorite and a suitable fluorescent dye with excellent optical properties are highly sought

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simultaneously. It is reported that the epoxidation reaction of α,β-unsaturated ketone

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with hypochlorite could occur at aqueous solution and room temperature to afford

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epoxide (Scheme 1A) [29-30]. We envisioned that this reaction may be exploited for designing a new ratiometric hypochlorite probe. On the other hand, the extremely

fluorescent

probe

with

large

emission

shift.

With these

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ratiometric

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long or short emission wavelength of the fluorescent dye is essential for constructing

considerations in mind, we associated the benzopyrylium-coumarin system (CBP),

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which possesses unsaturated structure that potentially can be used as a reaction site

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for hypochlorite, and showed extremely long emissionwavelength (generally > 650 nm) simultaneously (Scheme 1B). Because the coumarin moiety contained in

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benzopyrylium-coumarin based dyes generally emit blue fluorescence, the

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interruption of the π-conjugated system in the dyes may release the the fluorescence of the coumarin moiety. It can satisfy the need for constructing ratiometric fluorescent probes with large emission shift. The compound CBP was synthesized by the condensation

reaction

of

4-diethylamino-2-hydroxy-benzaldehyde

and

3-acetyl-7-hydroxy-chromen-2-one, and the structure ofCBP was characterized by 1H NMR, 13C NMR and HRMS spectra.

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ACCEPTED MANUSCRIPT

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Scheme 1. Strategy for the design of ratiometric fluorescent probe with large emission shift for

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detecting hypochlorite.

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3.2. Optical properties and theoretical calculations of the free probe CBP. Initially, we evaluated the optical properties of CBP in PBS (pH = 7.4, 20 mM, 5%

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EtOH) using UV-Vis absorption and fluorescence spectra (Fig. S1). The probe CBP

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itself exhibited a main absorption band centered at 628 nm with the molar extinction coefficient (ε) of 5.6 × 104 M-1 cm-1, and three relative weak absorption bands located

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at 260, 383 and 453 nm, respectively. When excited at 410 nm, CBP showed two emission bands centered at 662 and 456 nm, respectively. The emission at 662 nm

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could be ascribed to the large π-system made of the chromenylium and coumarin units. As for the emission at 456 nm, it may be explained by the structural change of CBP in aqueous solution (Fig.S2) [31]. The hydration reaction to chromenylium core, as well as the followed tautomerization could interrupt the large π conjugated system of CBP, and release the blue emission of the coumarin fluorophore. Density functional theory (DFT) calculations using a suite of Gaussian 09 programs 8

ACCEPTED MANUSCRIPT were performed to get insight into the optical properties of CBP. As shown in Fig. 1 and Fig. S4, the calculation results show that the chromenylium core is essentially coplanar and conjugated with coumarin moiety to afford a large π-system, which can be responsible for the absorption and emission bands at long wavelength. TD-DFT

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calculations indicated that the electronic transition is mainly contributed to

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HOMO-LUMO transition (Table S1). The π electrons of HOMO are mainly located at

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chromenylium core, while the π electrons of LUMO are primarily distributed over coumarin moieties at ground state and excited state. It indicates that intromolecular

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charge transfer (ICT) from chromenylium core to coumarin moiety occurs when CBP

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was excited. In addition, the TD-DFT calculation shows that CBP exhibits main

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fluorescence at 633 nm, which is close to the experimental value.

Fig.1 Frontier molecular orbital plots of CBP involved in the vertical excitation (i.e., UV/Vis absorption, left column) and emission (right column). The vertical excitation related calculations are based on the optimized geometry of the ground state (S0), and the emission related calculations

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ACCEPTED MANUSCRIPT were based on the optimized geometry of the excited state (S1). In the ball-and-stick representation, carbon, nitrogen and oxygen atoms are colored in gray, blue, red and yellow, respectively.

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3.3. Optical response of CBP to hypochlorite. To determine the optical response of CBP to hypochlorite, the UV-Vis spectra of CBP

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titrated with various concentrations of NaClO in PBS (pH = 7.4, 20 mM, 5% EtOH)

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were performed. In this work, NaClO was selected as hypochlorite source. Upon the

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addition of NaClO, CBP displayed a marked decrease in the absorption at 662 nm (Fig. 2). Meanwhile, the absorbance of the other three bands at 260, 383 and 453 nm

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decreased simultaneously with the addition of NaClO. This phenomenon indicates that the chemical structure of CBP changes after the treatment with NaClO. In

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additon, CBP solution displayed blue color under natrual light, and turned light after the addition of NaClO, consisting with the optical response of CBP to NaClO in

0.3

Absorbance

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UV-Vis spectra.

0.2

0.1

0.0

300

400

500

600

700

Wavelength / nm

Fig. 2 Absorption spectra of 5 μM CBP upon the incubation with increasing concentrations of NaClO (0-100 equiv) for 5 min in PBS (pH=7.4, 20 mM, 5% EtOH). The arrows indicate the change of the absorbance with the increase of NaClO from 0 to 100 equiv. Inset: The photographs 10

ACCEPTED MANUSCRIPT of 5 μM CBP in absence (left) and presence (right) of 100 μM NaClO under natrual light.

Next, the fluorescence spectra of CBP in presence of NaClO in PBS (pH=7.4, 20 mM, 5% EtOH) were performed. The emission intensity at 456 nm increased

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obviously with the enhancement of NaClO concentration, while the emission intensity at 662 nm decreased gradually (Fig.3). When irradiated by 365 nm ultraviolet

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radiation, CBP gave off strong red fluorescence in absence of NaClO, and emitted

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strong blue fluorescence in presence of NaClO, consisting with the fluorescence

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response of CBP toward NaClO. Notably, when CBP responded to NaClO, the extremely large hypsochromic shift of 206 nm in the emission spectra was observed,

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which could result in the well-separated emission peaks and decrease the fluorescence detection errors resulted from the spectral overlap between the probe and the product

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after responding to analyte. A linear correlation between the fluorescence intensity

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ratio I456/I662 and NaClO concentrations in the range of 0-50 μM was obtained (Fig.

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S5), and the detection limit was calculated to be 51 nM according to IUPAC recommendations (S/N= 3). Therefore, the probe CBP could serve as a potential

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ratiometric fluorescence probe with extremely large emission shift forsensing hypochlorite in living system.

(B)

1.2

100

0.4

0.0

50

600

0.8

I456/I662

Fl. Intensity

Fl. Intensity

(A) 150

650

700

750

400

200

Wavelength / nm

0

0 500

600

700

800

Wavelength / nm

0

100

200

300

400

Concentration/

11

500

ACCEPTED MANUSCRIPT Fig. 3 Fluorescence spectra (A) and the fluorescence intensity ratio I456/I662 (B) of 5 μM CBP incubated with NaClO (0-100 equiv.) for 5 min, λex = 410 nm. Insets in (B) are the photographs of 5 μM CBP in absence (left) and presence (right) of 100 μM NaClO at 365 nm ultraviolet

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radiation.

To confirm that the fluorescence response mechanism of CBP to hypochlorite was

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resulted by the hypochlorite-mediated epoxidation, the reaction product of CBP with

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NaClO (50 equiv) in PBS for 3 h was characterized by MS analysis. As shown in Fig.

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S6, the prominent peaks at m/z 362.1 and 396.1 corresponding to CBP (calculated M+ 362.1) and the proposed oxidative product CBP-ClO (calculated [M+H]+ 396.1) can

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be clearly observed. According this data and the above-mentioned optical response of CBP toward hypochlorite, we speculated that CBP may undergo hydration and

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tautomerization to provide ring-open product, and subsequently the double bond of the ring-open product reacts with hypochlorous acid to form chlorohydrin and then following intramolecular cyclization to form epoxide (Fig. S7).

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Subsequently, the time-dependent fluorescence spectra of CBP (5 μM) toward

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NaClO (100 μM) in PBS (pH=7.4, 20 mM, 5% EtOH) were performed to investigate the sensitivity of the probe. As shown in Fig. 4A, upon the addition of NaClO, the fluorescence intensities at 456 nm increased significantly with time, while the fluorescence intensities at 662 nm showed decrease. An obvious increase in the fluorescence ratio I456/I662 was observed after 1 min, and the ratio I456/I662 reached maximum within 4 min (Fig.4B). These results suggest that the probe CBP shows a rapid response to HOCl and can potentially serve as a retiometric probe for for 12

ACCEPTED MANUSCRIPT real-time imaging hypochlorite in biological system.

45

(B) 150

0.8

120

0.4 0.0

30

650

700

750

90 60

Wavelength / nm

30

15

0 0

500

600

700

800

0

9

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Fl. Intensity

60

1.2

I456/I662

Fl. Intensity

(A) 75

18

27

36

45

Time / min

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Wavelength / nm

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Fig. 4 Time-dependent fluorescence spectra (A) and fluorescence ratio (B) of 5 μM CBP in the

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presence of 100 μM NaClO in PBS (pH=7.4, 5% EtOH) under excitation at 410 nm.

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To evaluate the specificity of CBP to hypochlorite, the probe CBP (5 μM) was treated with a wide array of the representative species such as reactive

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oxygen/nitrogen species (ROS/RNS), cysteine, GSH in PBS (pH = 7.4, 20 mM, 5%

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EtOH). As shown in Fig.5, NaClO could simultaneously elicit the obviously enhanced fluorescence at 456 nm and the decreased fluorescence at 662 nm, along with the

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large ratiometric fluorescence ratio of I456/I662 up to 120, corresponding to the obvious fluorescent colorchange from red to blue after the addition of NaClO. Although

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Na2SO3 and Na2S also can elicit the obvious changes of the ratiometric fluorescence ratios of I456/I662, however, they cannot elict the strikingly incread fluorescence at 456 nm as NaClO. Namely, H2S and SO2 could disturb the ratiometric determination of HClO in biological systems, while they had less disturbance to the detection of HClO using the fluorescence signal at 456 nm. Meanwhile, the other ROS and RNS species elicited a weak ratiometric response with I456/I662 less than 3.0, indicating that CBP 13

ACCEPTED MANUSCRIPT has desirable selectivity for hypochlorite over the other related species.

(B)

1.2

CBP + NaClO

CBP + Na2SO3

15

CBP + Na2S

650

700

750

800

Wavelength / nm

CBP + Na2S CBP + other analytes

600

13, H2O2; 14, NaClO4; 15, NaClO; 16, Na2S; 17, Na2SO3.

60 30

0 500

9, TBHP; 10, VC; 11, NO; 12, NaNO2;

90

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0.0

30

CBP + Na2SO3

0.4

1, blank; 2, GSH; 3, Cys; 4, Hcy; 3+ 5, .OH; 6, ONOO ; 7, Fe ; 8, DTBP;

120

CBP + NaClO

700

800

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Wavelength / nm

0

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45

0.8

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Fl. Intensity

Fl. Intensity

60

150

CBP + other analytes

I456/I662

(A) 75

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Fig. 5 Fluorescence spectra (A) and fluorescence ratio I456/I662 (B) of 5 μM CBP incubated with

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various species for 5 min. The concentration of GSH was 1 mM, and the concentrations of other analytes were100 μM. Data were acquired in PBS (pH=7.4, 20 mM, 5% EtOH) with λex = 410 nm.

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The ratiometric fluorescence responses of the probe CBP (5 μM) to hypochlorite at

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various pH values were investigated as well. In the absence of NaClO, CBP itself showed a gradual increase for the fluorescence intensities at 456 and 662 nm in the

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pH range of 4-11 (Fig. S8). However, the emission ratio (I456/I662) of CBP exhibited a

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slight variation from 0.65 at pH 4 to 1.10 at pH 11 (Fig. S9). In the presence of NaClO (40 equiv), CBP exhibited an obvious increase of the fluorescence ratio (I456/I662) from 16 at pH 4 to 189 at pH 11, suggesting CBP showed more drastic response for hypochlorite at base condition (Fig. S8B and Fig. S9). The main reason for the increase of the ratio of I456/I662 is because that the I456 of the product is increased from pH 4 to pH11 (Fig S8B) similar to what was observed for CBP (Fig S8A), while the I662 remain at a low level (Fig S8B). Meanwhile, at higher pH, the 14

ACCEPTED MANUSCRIPT hydroxyl group is deprotonated. Since phenol anion is a better electron-donating group, the absorbance is increased along with the fluorescence emission intensity. At pH 7.4, the probe CBP exhibited a large fluorescence ratio (I456/I662) of 90, indicating that the probe CBP can be suitable for biological applications at the physiological pH

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3.4. Fluorescence imaging of cellular hypochlorite.

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value.

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Encouraged by the above-mentioned promising ratiometric responses of CBP to

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hypochlorite, we tested its biological application for the ratiometric imaging of hypochlorite in living cells. Initially, MTT assay was performed to investigated the

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cytotoxicity of CBP to the living HeLa cells. The results indicated that the low concentration of CBP had no marked cytotoxicity to the living HeLa cells (Fig. S10).

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Only incubated with 5 μM CBP for 30 min, HeLa cells showed nearly no blue fluorescence andstrong red fluorescence (Fig.6). When treated with CBP and then

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further incubated with NaClO (100 μM) for another 30 min, HeLa cells exhibited fluorescence in blue channel. Meanwhile, the red fluorescence intensity had slight

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decrease relative to the cells only treated with CBP. It suggests that CBP is cell membrane permeable and capable of ratiometric fluorescence imaging of hypochlorite in living cells.

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ACCEPTED MANUSCRIPT Bright field

Blue channel B1

Red channel C1

Merged D1

Ratio E1

CBP

A1

2.0

1.0

B2

C2

D2

E2

2.0

1.0

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CBP + NaClO

0

A2

0

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Fig. 6 (A1-E1) Images of Hela cells incubated with 5 μM CBP for 30 min. (A2-E2) Images of

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Hela cells incubated with 5 μM CBP for 30 min and then incubated with 100 μM NaClO for

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another 30 min. Blue channel: Excitation at 405 nm, emission window of 425-475 nm; red

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channel: Excitation at 647 nm, emission window of 663-738 nm. Scale bar: 20 μm.

4. Conclusions

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In conclusion, we constructed a novel ratiometric fluorescent hypochlorite probe

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based on the hypochlorite-mediated epoxidation reaction. The optical propeties of CBP was rationalized using DFT calculaitions. CBP showed high sensitivity and

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slectivity to hypochlorite. Importantly, CBP exhibited extremely large emission shift

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(206 nm) in response to hypochlorite, and could be benefit for the precise measurement of the fluorescence peak intensities and ratios. The biological imaging results demonstrated that CBP could be employed for ratiometric fluorescence imaging of hypochlorite in living cells. We expect that the hypochlorite-promoted epoxidation reaction could be widely used for the design of ratiometric fluorescent hypochlorite probe with large emission shift.

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ACCEPTED MANUSCRIPT Acknowledgement This work was financially supported by NSFC (21472067, 21672083, 51602127), Taishan Scholar Foundation (TS 201511041), and the startup fund of the University of

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Jinan (309-10004).

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ACCEPTED MANUSCRIPT [29] B. Lygo, P.G. Wainwright, Tetrahedron Lett. 39(1998)1599-1602. [30] B. Lygo, P.G. Wainwright, Tetrahedron, 30(1999)6289–6300. [31] A. Roque, C. Lodeiro, F. Pina, M. Maestri, S. Dumas, P. Passaniti, V. Balzani, J.

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Am. Chem. Soc.125(2003)987-994.

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ACCEPTED MANUSCRIPT

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Graphical Abstract

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ACCEPTED MANUSCRIPT Highlight: (1) Utilizing positively charged α,β-unsaturated carbonyl group as the new reaction site, the probe CBP showed high sensitivity and selectivity for hypochlorite based on epoxidation reaction of α,β-unsaturated ketone.

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(2) When responded to hypochlorite, CBP showed an extremely large emission shift

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of 206 nm, which far exceeded most of the developed ratiometric fluorescent

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hypochlorite probe.

(3) DFT calculations were performed to provide insights into the structure-optical

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properties of the probe CBP.

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(4) The probe CBP has been successfully applied for the ratiometric imaging of

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hypochlorite in living cells.

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