A hydrazone-based turn-on fluorescent probe for peroxynitrite detection and live-cell imaging

Journal Pre-proof A hydrazone-based turn-on fluorescent probe for peroxynitrite detection and live-cell imaging Sudeok Kim, Chang Woo Ko, Taeho Lim, Soyeon Yoo, Hye Jin Ham, Seo-Young Kang, Seungyoon Kang, Steve K. Cho, Min Su Han PII:

S0143-7208(19)31246-X

DOI:

https://doi.org/10.1016/j.dyepig.2019.107762

Reference:

DYPI 107762

To appear in:

Dyes and Pigments

Received Date: 30 May 2019 Revised Date:

29 July 2019

Accepted Date: 30 July 2019

Please cite this article as: Kim S, Ko CW, Lim T, Yoo S, Ham HJ, Kang S-Y, Kang S, Cho SK, Han MS, A hydrazone-based turn-on fluorescent probe for peroxynitrite detection and live-cell imaging, Dyes and Pigments (2019), doi: https://doi.org/10.1016/j.dyepig.2019.107762. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier Ltd.

Graphical Abstract

1

A hydrazone-based turn-on fluorescent probe for peroxynitrite detection

2

and live-cell imaging

3 4

Sudeok Kima, Chang Woo Kob, Taeho Lima, Soyeon Yooa, Hye Jin Hamc, Seo-Young Kangd, Seungyoon

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Kanga, Steve K. Chob and Min Su Hana,*

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a

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Korea

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b

Department of Chemistry, Gwangju Institute of Science and Technology (GIST), Gwangju 61005, Republic of

School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju 61005, Republic of

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Korea

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c

GIST College, Gwangju Institute of Science and Technology (GIST), Gwangju 61005, Republic of Korea

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d

International Environmental Research Institute (IERI), Gwangju Institute of Science and Technology (GIST),

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Gwangju 61005, Republic of Korea

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*Corresponding author. Tel.: +82 62 715 2850; Fax: +82 62 715 2866.

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E-mail address: [email protected] (M. S. Han)

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Abstract

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Peroxynitrite (ONOO–) plays an important physiological role and is related to various clinical diseases. In the

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past two decades, a number of fluorescent probes have been developed to detect ONOO–, however, most of

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them show insufficient selectivity over other reactive oxygen species (ROS) and reactive nitrogen species

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(RNS), long response time, low fluorescence enhancement in the presence of ONOO–, and poor water solubility.

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Herein, a hydrazone-based fluorescent probe that overcomes the abovementioned problems was designed and 1

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synthesized by a single-step reaction. This probe exhibits high sensitivity to ONOO–, with ~76-fold

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enhancement of fluorescence intensity within 1 minute, and excellent selectivity for ONOO– over various

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ROS/RNS. Furthermore, this probe was successfully applied for the visualization of ONOO– in RAW264.7 and

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H1299 cells.

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Key words: Peroxynitrite, Fluorescent probe, Hydrazone hydrolysis, Simple synthesis, Live-cell imaging

30 31

1. Introduction

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Peroxynitrite (ONOO–) is a highly reactive nitrogen species (RNS) that plays an important role in various

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physiological conditions [1,2]. ONOO– is formed from the reaction between the superoxide radical (O2•–) and

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nitric oxide (NO) [3]. An abnormal concentration of ONOO– has been associated with various clinical diseases,

35

such as inflammatory, cardiovascular, and Alzheimer’s diseases, and cancer [4,5]. In addition, because it can

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generate reactive oxygen species (ROS) and RNS by stimulation of immune cells, detection methods for

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ONOO– are very important in immunology research [6,7]. Therefore, it is crucial to develop an efficient method

38

for the detection of ONOO– in biological systems.

39

A fluorescent probe, an efficient tool for analytical methods, is having advantages of high sensitivity,

40

nondestructive detection and real-time monitoring [8-11]. In the past two decades, a number of fluorescent

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probes have been developed to detect ONOO– that are based on hydrazine or hydrazone [12,13], boronate

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[14,15], selenium or tellurium [16,17], polymethine [18,19], N-(4-hydroxyphenyl) amino [20-22], α-ketoamide

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[23], and other moieties [24-26]. However, most of them showed insufficient selectivity over other ROS/RNS,

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long response time, low fluorescence enhancement in the presence of ONOO– in physiological conditions, and

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poor water solubility, which greatly limited their biological applications. Therefore, the development of a

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fluorescent probe for ONOO– that addresses the above problems is still necessary.

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To develop an excellent probe for the detection of ONOO–, we chose a coumarin dye as a fluorophore due to its

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many advantages, such as being an easy-to-modify functional group with excellent photophysical properties

49

[27]. Using these properties, various hydrazine-selective fluorescent probes have been developed [28-30]. On 2

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the other hand, hydrolysis of hydrazones derivatives, as a common carbonyl protecting group [31], is known to

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be possible in the presence of oxidants [32-34]. Therefore, we designed a hydrazone-based turn-on fluorescent

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probe for peroxynitrite detection by combining 7-diethylamino-3-formylcoumarin (2), as a fluorophore, and

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dimethyl hydrazone, as a ONOO–-reactive group (Scheme 1).

54



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Here, we report the development of a simple and cost-effective fluorescent probe (1), via a single synthetic step

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for an excellent selective and sensitive detection of ONOO– in physiological conditions without the interference

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of other ROS/RNS.

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2. Experimental Section

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2.1. General

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All the materials were purchased from commercial suppliers (Sigma-Aldrich, Tokyo Chemical Industry, and

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Alfa Aesar) and were used without further purification. 1H and 13C NMR spectra were obtained using a JEOL

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ECS 400 MHz NMR spectrometer. An Agilent ESI-Q/TOF (quadrupole/time-of-flight) mass spectrometer was

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used to obtain mass spectra. Melting points were measured using a BUCHI melting point M-565 apparatus.

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Fluorescence spectra were recorded with an Agilent Cary Eclipse fluorescence spectrometer. UV-Vis

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spectrometry was carried out on a JASCO V-630 spectrophotometer. UPLC-MS analyses were performed on a

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Waters Acquity I-class system equipped with a PDA detector (UPLC eLambda 800 nm) coupled to a Waters

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Quattro micro API. Fluorescence imaging of cells was carried out in an Olympus FV1000 confocal laser

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scanning microscope (CLSM).

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2.2. Synthesis of 7-(diethylamino)-2-oxo-2H-chromene-3-carbaldehyde (2)

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The synthetic procedure of 2 followed the literature [35]. 1H NMR (400 MHz, CDCl3): δ = 10.13 (s, 1H), 8.25 (s,

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1H), 7.41 (d, J = 8.9 Hz, 1H), 6.64 (dd, J = 8.9, 2.5 Hz, 1H), 6.49 (d, J = 2.5 Hz, 1H), 3.48 (q, J = 7.1 Hz, 4H),

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1.26 (t, J = 7.1 Hz, 6H).

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2.3. Synthesis of (E)-7-(diethylamino)-3-((2,2-dimethylhydrazineylidene)methyl)-2H-chromen-2-one (1)

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Compound 2 (0.49 g, 2.0 mmol) and 1,1-dimethylhydrazine hydrochloride (0.39 g, 4.0 mmol) were mixed in 3

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ethanol (80 mL) (Scheme 1). The solution was stirred at room temperature for 36 h. After the reaction was

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finished, ethanol was removed under reduced pressure. The residue was dissolved in chloroform and washed

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with water. The organic layer was dried with anhydrous sodium sulfate, filtered, and concentrated under reduced

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pressure. The crude product was recrystallized from ethanol to give product 1 (0.32 g, 1.1 mmol), with 56 %

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yield. mp 110-112°C; 1H NMR (400 MHz, CDCl3): δ = 8.01 (s, 1H), 7.35 (s, 1H), 7.29 (d, J = 8.9 Hz, 1H), 6.57

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(dd, J = 8.9, 2.4 Hz, 1H), 6.50 (d, J = 2.4 Hz, 1H), 3.41 (q, J = 7.0 Hz, 4H), 2.99 (s, 6H), 1.21 (t, J = 7.2 Hz, 6H);

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13

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12.5; HRMS (ESI): [M + H]+ m/z calculated for C16H22N3O2+: 288.1707; found: 288.1708.

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2.4. Preparation of various ROS, RNS, and biothiols

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ONOO– was synthesized using a modified procedure described by Reed et al [36]. Briefly, a mixture of sodium

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nitrite (NaNO2, 0.6 M) and hydrogen peroxide (H2O2, 0.7 M) was acidified with hydrochloric acid (HOCl, 0.6

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M), and sodium hydroxide (NaOH, 1.5 M) was added within 1-2 s to make the solution alkaline. The

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concentration of ONOO– was determined by measuring the absorption of the solution at 302 nm. The extinction

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coefficient of the peroxynitrite solution in 0.1 M NaOH is 1670 M-1 cm-1, at 302 nm [37]. Hydrogen peroxide is

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commercially available; the concentration of H2O2 was determined through spectrophotometrical analysis with ε

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= 43.6 M-1 cm-1, at 240 nm [38]. The source of HOCl was commercial bleach. The concentration of HOCl was

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determined through spectrophotometrical analysis. The extinction coefficient of the HOCl solution in 0.1 M

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NaOH is 350 M-1 cm-1, at 292 nm [39]. tert-Butyl hydroperoxide (TBHP) was prepared in deionized distilled

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water by diluting 70% TBHP. ROO• was generated from 2,2’-Azobis(2-amidinopropane) dihydrochloride,

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which was firstly dissolved in deionizer water [40]. The hydroxyl radical (HO•) was generated by the Fenton

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reaction [41]. To generate HO•, ferrous chloride was added in the presence of 10 equivalents of H2O2. The

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concentration of HO• was equal to that of Fe(II) concentration. 1O2 was chemically generated from the

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H2O2/NaOCl system in physiological media [42]. The superoxide solution (O2•–) was prepared by adding KO2 to

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dry dimethyl sulfoxide (DMSO) and stirring vigorously for 10 min [43]. Nitric oxide was generated using

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sodium nitroferrycyanide (SNP). NO2– and NO3– were prepared by dissolving NaNO2 and NaNO3 in deionized

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distilled water, respectively. Cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) were prepared by

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dissolving L-cysteine, DL-homocysteine, and L-glutathione in deionized distilled water, respectively.

C NMR (100 MHz, CDCl3): δ = 162.5, 155.7, 150.1, 134.1, 129.1, 126.8, 116.9, 109.3, 109.1, 97.3, 44.8, 42.9,

4

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2.5. Sensitivity of ONOO– detection by 1

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Various concentrations of ONOO– (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12.5, 15, 17.5, and 20 µM) were added to

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sodium phosphate buffer (SPB, 0.1 M, pH 7.4) containing 1 (1 µM, 1% DMSO). Fluorescence spectra were

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obtained after 1 min. λex = 440 nm. Slit widths: ex = 10 nm; em = 10 nm.

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2.6. Selectivity of 1 toward ONOO– over other ROS/RNS/biothiols

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Various ROS/RNS/biothiols (20 µM H2O2, TBHP, HOCl, ROO•, HO•, 1O2, O2•–, NO, NO2–, NO3–, Cys, Hcy,

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GSH, and 10 µM ONOO–) were added to SPB containing 1 (1 µM, 1% DMSO). The fluorescence spectra were

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obtained after 1 min. λex = 440 nm. Slit widths: ex = 10 nm; em = 10 nm.

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2.7. Mechanistic study of ONOO– detection by 1

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2.7.1. Preparation of samples

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Various concentrations of ONOO– (0, 0.5, 1, 2, 5, 10, and 20 µM) were added to SPB containing 1 (1 µM, 1%

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DMSO) with 10 volumes scales (final volume: 10 mL). After 5 min, ethyl acetate (EA, 10 mL) was added to the

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reaction mixture. The organic layer was concentrated under reduced pressure. Finally, the residue was dissolved

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in acetonitrile (1 mL).

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2.7.2. UPLC-MS analytical methods

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For the analyses, an Acquity UPLC BEH-C18 column (1.7 µm, 2.1 mm × 50 mm) was used with UPLC grade

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water (A) and acetonitrile (B) as eluents (both containing 0.1% formic acid). The UV absorbance was monitored

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at 465 nm, and the column temperature was kept at 40°C. The injection volume was 5.0 µL (flow 10 µL min-1).

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The elution method is shown in Table S1. The mass spectrometer was operated in positive ES mode, with

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capillary, cone, and extractor voltages kept at 3 kV, 20 V, and 3 V, respectively. The source and desolvation

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temperatures were set 150°C and 350°C, respectively. The cone gas flow was 30 L h-1, and the desolvation gas

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flow was 500 L h-1.

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2.8. pH dependence

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ONOO– (10 µM) was added to various buffer solutions (0.1 M, pH 4 - 10) containing 1 (1 µM, 1% DMSO). The

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fluorescence spectra were measured after 1 min. λex = 440 nm. Slit widths: ex = 10 nm; em = 10 nm. The 5

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compositions of the buffer solutions were as follows: pH 4 - 5 (sodium acetate), pH 6 - 8 (sodium phosphate),

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pH 9 - 10 (CHES).

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2.9. Cell culture

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RAW264.7 (murine macrophage) cells and H1299 (human lung carcinoma) cells were obtained from the Korean

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Cell Line Bank (Seoul, Korea). RAW264.7 cells were cultured in Dulbecco’s Modified Eagle’s Medium and

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H1299 cells were cultured in RPMI 1640 Medium (Gibco, Gland Island, NY, USA), respectively, supplemented

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with 100 U mL-1 penicillin, 100 µg mL-1 streptomycin, and 10% fetal bovine serum. All cells were kept in a

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humidified atmosphere with 5% CO2, at 37°C.

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2.10. Cell viability assay

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Cells were seeded in a 96-well plate (104/well) with culture medium. After 24 h incubation, the culture medium

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was replaced, and the cells were incubated with various concentrations of 1 (0, 1, 2, 5, 10, 20, 50, 100, and 200

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µM) for 24 h. Ten microliters of MTT solution (0.5 mg mL-1, DPBS) were, then, added to each well. After 4 h,

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100 µL of DMSO were added to each well to dissolve the formazan crystals. The absorbance was measured at

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570 nm in a CYTATION 3 microplate reader.

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2.11. Confocal microscope imaging

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Cells were seeded in 35-mm glass bottom dishes at a density of 2 × 105 cells per dish in culture medium. After

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24 h, the culture medium was removed, and the cells were incubated with 50 µM of 1 for 1 h, washed with

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Dulbecco’s phosphate buffered saline (DPBS). And the cells were treated with 200 µM 5-amino-3-(4-

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morpholinyl)-1,2,3-oxadiazolium chloride (SIN-1) in the absence or presence of uric acid (50 µM) for 1 h.

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Finally, fluorescence images were acquired by confocal laser scanning microscopy (FV1000, Olympus, Japan)

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in Hank’s balanced salt solution (HBSS). λex = 440 nm; λem = 460 - 560 nm.

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3. Results and Discussion

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Spectroscopic evaluation of 1 was performed under physiological conditions (0.1 M SPB, pH 7.4). Firstly, the

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corresponding fluorescence spectra were measured with an excitation wavelength of 440 nm. 1 was

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nonfluorescent in physiological conditions. However, upon addition of 10 equivalents of ONOO–, the

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fluorescence intensity of 1 at 480 nm increased rapidly and reached a plateau in a short period of time (1 min, 6

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Fig. S11). Consequently, a remarkable enhancement in fluorescence (~76-fold) at 480 nm could be observed

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(Fig. 1). Moreover, an excellent linear relationship was obtained over an ONOO– concentration ranging from 0

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to 10 µM, and the detection limit was estimated to be 35 nM (Fig. S5). These results indicate that 1 can be used

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for quantitative detection of ONOO– with high sensitivity.

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To evaluate the selectivitiy of 1 toward ONOO–, its reactivity with various ROS/RNS/biothiols was tested. As

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shown in Fig. 2, a large enhancement in fluorescence intensity was observed with ONOO– (10 µM), while other

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analytes (20 µM) could only give a negligible fluorescence increase for 30 min (Fig. S11). Compared with

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previously reported fluorescent probes for ONOO–, 1 rarely showed the interference caused by other ROS/RNS,

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such as H2O2, HOCl, HO•, O2•–, and NO [44-48]. On the other hand, 1 exhibited an absorption band at 436 nm

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(Fig. S12). Upon addition of ONOO–, the absorption was shifted to 424 nm, whereas addition of other

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ROS/RNS/biothiols showed no effect to the change of absorption band. These results demonstrate that 1 has

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excellent selectivity for ONOO–, which enables it to selectively detect ONOO– in physiological conditions. This

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feature makes 1 more advantageous than most of the previously reported fluorescent probes for ONOO–.

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In order to verify the proposed mechanism, ultra performance liquid chromatography/mass spectroscopy

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(UPLC/MS) analysis was employed. The spectrum of 1 displayed a signal peak at 7.65 min, and this peak,

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corresponding to [1 + H]+, appeared at m/z = 288.12 (Fig. S6). Upon adding ONOO–, the decrease in the signal

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of the probe was observed along with the appearance of new signals at 6.34 min, which are attributed to the

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product. When 10 equivalents of ONOO– were added to the solution, a peak corresponding to [2 + H]+ appeared

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at m/z = 246.12, demonstrating the complete hydrolysis of 1 (Fig. S7). These results are consistent with the

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proposed mechanism (Fig. 3).

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Also the effect of pH on the fluorescence enhancement in the presence of ONOO– was evaluated in a pH range

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of 4 – 10 (Fig. 4). In the absence of ONOO–, the fluorescence intensity of 1 exhibited weak fluorescence at a pH

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range of 5 - 10. On the other hand, upon addition of ONOO–, fluorescence intensities were increased in a pH 7

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range of 5 - 9. In particular, strong fluorescence signals were observed in the pH range 7 - 7.4, which implies 1

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could be used as a fluorescent probe for ONOO– in physiological conditions. Meanwhile, to verify the

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interference of metal ions, we added metal ions which are present in biological fluids followed by addition of

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ONOO– [49]. Metal ions did not significantly affect fluorescence responses of 1 toward ONOO– (Fig. S13).

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Therefore, combined with the results of its response behavior, 1 is possible to be applied to biological imaging.

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First of all, we conducted experiment in RAW264.7 cells, which are murine macrophage cells of the immune

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system. Before confocal laser scanning microscope (CLSM) imaging, the cytotoxicity of 1 in living cells was

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assessed by the MTT assay (Fig. S8). RAW264.7 cells were incubated with increasing concentrations of 1 for 24

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h. 1 showed negligible cytotoxicity below 50 µM, and 1 was used at the concentration that did not affect cell

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viability. Then, its potential for fluorescent detection of exogenous ONOO– in RAW264.7 cells was evaluated

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(Fig. 5). Cells incubated only with 1 showed negligible fluorescence in the cyan channel (Fig. 5b). When cells

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were treated with SIN-1 (an ONOO– donor), however, a strong fluorescence enhancement in the cyan channel

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was observed (Fig. 5c). In addition, when the cells were pretreated with 50 µM uric acid, a well-known ONOO–

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scavenger [50], the fluorescence intensities of the images effectively decreased to basal levels (Fig. 5d). Also,

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we performed experiment in H1299 cells, that are human carcinoma cells. The fluorescent detection of

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exogenous ONOO– in H1299 cells was performed successfully (Fig. S10). These results imply that 1 has the

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capacity to observe exogenous ONOO– in cancer cells as well as immune cells.

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4. Conclusions

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In summary, we developed a hydrazone-based turn-on fluorescent probe, 1, for sensitive and selective detection

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of ONOO–, by combining 7-diethylamino-3-formylcoumarin, as a fluorophore, and dimethyl hydrazone, as the

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ONOO–-reactive group. This fluorescent probe possesses an excellent selectivity and sensitivity toward ONOO–

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over other ROS/RNS, a large fluorescent turn-on signal, and a fast response. Furthermore, the probe can be used

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to visualize ONOO– levels in RAW264.7 and H1299 cells with negligible background fluorescence and cellular

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toxicity.

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Acknowledgements

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This research was a part of the project titled ‘Smart bio sensing technology for managing distribution and safety

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about fishery products and processed fishery products’, funded by the Ministry of Oceans and Fisheries, Korea,

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and this work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean

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government (MSIT) (NRF-2017R1A2B4009652 and NRF-2018R1A4A1024963).

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Appendix A. Supplementary data

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Supplementary data related to this article can be found at doi ****

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Figure, Scheme, Table captions

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Fig. 1. (a) Fluorescence spectra of 1 (1 µM) with several concentrations of ONOO–. (b) Fluorescence intensities

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of 1 at 480 nm upon addition of ONOO– (0 - 20 µM). Each spectrum was recorded after 1 min.

332 333

Fig. 2. (a) Fluorescence spectra and (b) fluorescence responses of 1 (1 µM) toward ONOO– (10 µM) and other

334

ROS/RNS/biothiols (20 µM) in SPB (0.1 M, pH 7.4, containing 1% DMSO) after 1 min. λex = 440 nm.

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Fig. 3. (a) Proposed sensing mechanism of ONOO– by 1. (b) UPLC/MS chromatograms of the reaction between

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1 and ONOO–. The peak signals were detected by monitoring the absorbance at 465 nm.

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Fig. 4. Fluorescence intensity at 480 nm of 1 (1 µM) in the absence or presence of ONOO– (10 µM) at varied pH

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values. λex = 440 nm.

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Fig. 5. Live-cell imaging of 1 by confocal laser scanning microscopy. (a) RAW264.7 cells only. (b) RAW264.7

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cells loaded with 50 µM 1 for 1 h. (c) 1-loaded cells were treated with 200 µM SIN-1 for 1 h. (d) 1-loaded cells

344

were pretreated with 50 µM uric acid followed by treatment with 200 µM SIN-1 for 1 h. The first and the

345

second rows show fluorescence images in the cyan channel and bright field images, respectively. ex = 440 nm;

346

em = 460 - 560 nm. Scale bar: 10 µm.

347 348

Scheme 1. Schematic illustration of a hydrazone-based turn-on fluorescent probe for ONOO–.

14

Fig. 1. (a) Fluorescence spectra of 1 (1 µM) with several concentrations of ONOO–. (b) Fluorescence intensities of 1 at 480 nm upon addition of ONOO– (0 - 20 µM). Each spectrum was recorded after 1 min.

Fig. 2. (a) Fluorescence spectra and (b) fluorescence responses of 1 (1 µM) toward ONOO– (10 µM) and other ROS/RNS/biothiols (20 µM) in SPB (0.1 M, pH 7.4, containing 1% DMSO) after 1 min. λex = 440 nm.

Fig. 3. (a) Proposed sensing mechanism of ONOO– by 1. (b) UPLC/MS chromatograms of the reaction between 1 and ONOO–. The peak signals were detected by monitoring the absorbance at 465 nm.

Fig. 4. Fluorescence intensity at 480 nm of 1 (1 µM) in the absence or presence of ONOO– (10 µM) at varied pH values. λex = 440 nm.

Fig. 5. Live-cell imaging of 1 by confocal laser scanning microscopy. (a) RAW264.7 cells only. (b) RAW264.7 cells loaded with 50 µM 1 for 1 h. (c) 1-loaded cells were treated with 200 µM SIN-1 for 1 h. (d) 1-loaded cells were pretreated with 50 µM uric acid followed by treatment with 200 µM SIN-1 for 1 h. The first and the second rows show fluorescence images in the cyan channel and bright field images, respectively. ex = 440 nm; em = 460 - 560 nm. Scale bar: 10 µm.

Scheme 1. Schematic illustration of a hydrazone-based turn-on fluorescent probe for ONOO–.

Highlights

A hydrazone-based turn-on fluorescent probe for peroxynitrite (ONOO–) was developed. This probe exhibits high sensitivity, excellent selectivity, and a rapid response time. This probe was successfully applied to the imaging of exogenous ONOO– in living cells.