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A Highly Selective and Sensitive Two-Photon Fluorescence Probe for Endogenous Peroxynitrite Detection and Its Applications in Living cells and Tissues Jun Li, Chang Su Lim, Gyoungmi Kim, Hwan Myung Kim, and Juyoung Yoon Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02059 • Publication Date (Web): 11 Jul 2017 Downloaded from http://pubs.acs.org on July 11, 2017

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Analytical Chemistry

A Highly Selective and Sensitive Two-photon Fluorescence Probe for Endogenous Peroxynitrite Detection and Its Applications in Living Cells and Tissues Jun Li,†, ‡ Chang Su Lim,§, ‡ Gyoungmi Kim,† Hwan Myung Kim,*,§ Juyoung Yoon*,† †

Department of Chemistry and Nano Science, Ewha Womans University, Seoul 120-750, Korea, [email protected], fax: 82-2-3277-2385. §

Department of Chemistry and Energy Systems Research, Ajou University, Suwon, Korea, [email protected], fax: 82-31-219-1615. ABSTRACT: A new two-photon fluorescence probe for endogenous peroxynitrite (ONOO-) detection was designed and synthesized. The probe exhibits good selectivity and sensitivity for ONOO- in phosphate-buffered saline solution with a low detection limit (3.5 ×10-8 M). Furthermore, the probe displays good performance in detecting endogenous ONOO-,not only in RAW 264.7 cells but also in rat hippocampal tissue, with a high two-photon cross-section value (δ ≈ 100 GM) at a deep depth of 120 μm.

INTRODUCTION Peroxynitrite (ONOO-) plays a crucial role in biological systems, and this has attracted broad interest from the scientific research community. In vivo, ONOO- is the . product of a reaction between the superoxide ( O2-) andnitric oxide (NO) free radicals, the latter of which is a powerful oxidant that can damage various molecules incells, includingDNAandproteins.1-3 Thus, abnormal levels of ONOO- in cells may be associated with diseases such as ischemia-reperfusion injury, inflammatory conditions, and neurodegenerative disease.4 Therefore, detecting ONOO- in biological systems may be useful in the early diagnosis of human disease. Fluorescence probesare efficient toolsfor the detection of biological molecules in vivo due to their high sensitivity and simplicity.5-8 Recently, considerable effort has been expended in developing ONOO- probes, and many of these probes exhibit good performance in live cells or mice.11-23 As such, some probes can also be revisable between glutathione (GSH) and ONOO-.7, 24, 25 However, despite the encouraging progress made to date, there remain considerable challenges with respect to developing fluorescent probes for ONOO-, including the high interference by HOCl and H2O2, a turnoff response, and a lack of tissue imaging.9, 10 Two-photon fluorescence probes utilize two near-infrared photons (700–900 nm), and possess many advantages compared with one-photon probes, including deeper tissue penetration (>70 nm) to overcome photo-bleaching and cellular auto-fluorescence.26-31 To date, few two-photon fluorescence probes have been utilized to image ONOO- in tissues.Very recently, however, Hu and Han et al. developed

a new two-photon ONOO- probe to visualize ONOOfluxes in endothelial cells, and used this to reveal the dynamic progression of vascular injury in the brain.32 Yuan et al. developed a ratiometric two-photon fluorescent probe to visualize endogenous ONOO- in mouse model of inflammation,33 whereas Tang’s group reported a naphthalimide-based two-photon fluorescent probe, which was used to reveal drug-induced hepatotoxicity via the mapping of ONOO- fluctuation.34 However, despite progress made in the development of two-photon fluorescent probes for ONOO-, certain aspects still need improving, including water solubility, two-photon cross-section action, and structural diversity. Herein, we report on a new two-photon fluorescence probe for endogenous ONOO- detection. This probe displays good water solubility and low cytotoxicity, thereby facilitating efficient detection of ONOO-under physiological conditions. Furthermore, the probe exhibits good performance in rat hippocampal tissue imaging, with a high two-photon cross-section value (δ ≈ 100 GM) at a deep depth of 120 μm (Table S-1).

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Scheme 1 Design and sensing mechanism of the ONOO probe.

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The design of the new probe, which involved an Ndearylation reaction with ONOO-, is depicted in Scheme 1. We envisioned that the fluorescence of the fluorophore could be quenched by the N-phenyl group, which is regarded as the reaction site of ONOO-. After the reaction with ONOO-, released product 1 containing a strong electron-donating group could exhibit strong fluorescence intensity with two-photon properties. The synthetic route and procedure are shown in the supporting information, and the target compound undergoes five steps with moderate yields. All compounds were confirmed and characterized via NMR and MS.

EXPERIMENTAL SECTION Materials and Chemicals Unless otherwise stated, all starting materials and reagents, including anhydrous dimethylformamide (DMF), dichloromethane (DCM), and triethylamine (Et3N), were obtained from commercial sources. 1H and 13C NMR spectra were measured in CDCl3 and dimethylsulfoxide (DMSO)-d6 solutions using a Bruker AM-300 spectrometer with tetramethylsilane (TMS) as the internal standard. Mass spectra were measured in electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI) mode. UV/Vis spectra were obtained using a Scinco 3000 spectrophotometer (1-cm quartz cell) at 25°C. Fluorescence spectra were recorded using an RF-5301/PC (Shimada) fluorescence spectrophotometer (1-cm quartz cell) at 25°C. Low-resolution LC/MS data were collected using a Hewlett-Packard series 1100 LC/MS system with a reversed-phase C18 column (Phenomenex Luna, 4.6 mm × 100 mm, 5 µm). Cell Culture RAW 264.7 cells (mouse macrophage cells; ATCC, Manassas, VA, USA) were cultured in Dulbecco’s modified Eagle medium (DMEM) (WelGeneInc., Seoul, Korea) supplemented with 10% fetal bovine serum (WelGene) and penicillin (100 units/ml).31 Cells were passed and plated in glass-bottomed dishes (NEST) before imaging for 2 days. Then, the cells were maintained in a humidified atmosphere of CO2/air (5%/95%, v/v) at 37°C. The growth medium was removed and replaced with serum-free DMEM for labeling.31 The cells were treated with 5 μM probe an incubated at 37°C under 5% CO2 for 20 min. Preparation of Reactive Oxygen Species (ROS) Solutions Hydrogen peroxide (H2O2), hypochlorite (ClO−), and tert-butyl hydroperoxide (TBHP) were obtained as aqueous solutions from commercial sources. Superoxide solution (O2−) was prepared by adding KO2 to dry DMSO and stirring vigorously for 10 min. Lipid peroxy radicals (ROO•)were generated from 2, 2'-azobis(2amidinopropane)dihydrochloride. Hydroxyl radicals (•OH) were generated by the Fenton reaction, and nitric oxide (NO) was used from a stock solution prepared from sodium nitroferricyanide (III) dihydrate (SNP). Peroxynitrite solution (ONOO-) was synthesized by a mixture of sodium nitrite and H2O2. The ONOO- concentration was estimated using an extinction coefficient of 1670 M−1cm−1 at 302 nm.

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Preparation and Staining of Fresh Rat Hippocampal Slices Rat hippocampal slices were prepared from the hippocampi of 2-week-old rats (Sprague–Dawley) according to an approved institutional review board protocol. Coronal slices were prepared at a thickness of 400 μm using a vibratome (Leica) in artificial cerebrospinal fluid (ACSF; 138.6 mM NaCl, 3.5 mM KCl, 21 mM NaHCO3, 0.6 mM NaH2PO4, 9.9 mM D-glucose, 1 mM CaCl2, and 3 mM MgCl2). Slices were labeled with the probe (20 μM) in ACSF bubbled with a gaseous mixture of 95% O2 and 5% CO2 for 1 h 30 min at 37°C. The slices were then washed three times by using ACSF, transferred to glass-bottomed dishes (NEST), and imaged under a spectral confocal multiphoton microscope. Two-photon Fluorescence Microscopy Two-photon fluorescence microscopy images of probe-labeled cells and tissues were obtained using spectral confocal multiphoton microscopes (Leica TCS SP8 MP) with ×10 dry and ×100 oil objectives and numerical apertures (NA) of 0.30 and 1.30, respectively.31 The fluorescence images of two-photon microscopy(DMI6000B Microscope, Leica) were collected by using a 750 nm and mode-locked titanium-sapphire laser source (Mai Tai HP; Spectra Physics, 80-MHz pulse frequency, 100-femto-second (fs) pulse width).The average power of laser source in the focal plane was approximately 15 mW. To obtain images in the 400–650 nm range, internal PMTs were used to collect the signals in 8-bit unsigned 512 × 512 and 1024 × 1024 pixels at 400- and 200-Hz scan speed,31 respectively. Measurement of Two-photon Cross-section The twophoton cross-section (δ) was determined using the fs fluorescence measurement technique as describedelsewhere.31, 35 The probe (1.0 × 10-6 M) was dissolved in phosphate-buffered saline (PBS) containing 1% DMF (10 mM, pH = 7.4) and the two-photon-induced fluorescence intensity was measured at 720−880 nm using rhodamine 6G as a reference, the two-photon property of which has been well characterized in the literature. 31, 35 The intensities of the two-photon-induced fluorescence spectra of the reference and sample emitted at the same excitation wavelength were determined. The two-photon absorption (TPA) cross-section was calculated using the following equation: δ = δr (SsФrφrcr)/(SrФsφscs), where the subscripts s and r are the sample and reference molecules, respectively, S is the intensity of the signal collected by a CCD detector, Φ is the fluorescence quantum yield,φ is the overall fluorescence collection efficiency of the experimental apparatus, c is the numerical density of the molecules in solution, and δr is the TPA cross-section of the reference molecule.31,35

RESULTS AND DISCUSSION Fluorescence Response of the Probe to Peroxynitrite. After obtaining the probe, we evaluated the photophysical properties of this probe (1 µM) for ONOO- in PBS solution (containing 1% DMF). The probe exhibited an obvious absorption peak at 370 nm, which was selected as the excitation wavelength (Figure S-1), and it showed very


weak fluorescence intensity (φ = 0.007). After the addition of 10 equivalences of ONOO-,the phenol group was oxidized to benzoquinone by ONOO-, accompanied by N−C bond cleavage (Scheme 1), and released product 1 displayed strong fluorescence intensity (φ = 0.1). No enhancement in the fluorescence was obtained with the other ROS solutions, including OCl-, H2O2, .O2-, NO, OH., ROO. and tBuOOH (Figure 1 and 2). Since many fluorophores and recognition sites are fragile to other ROS in addition to ONOO-, we further evaluated whether the probe would be adequate for ONOO- detection when coexisting with other ROS. As shown in Figure S-8, the newly designed probe exhibits excellent compatibility with other ROS, indicating that this probe could be used to detect and image endogenous ONOO-. The impact of pH is also regarded as an important factor in evaluating the stability of a fluorescent probe. Figure S-4 indicates that after the addition of four equivalences of ONOO-, our probe is very stable within the pH range 4 to 10.Furthermore,the increase in fluorescence intensity showed pH dependence, and the detection limit of the probe was determined to be 3.5 ×10-8 M (CDL = 3 Sb/m) (Figure S-3). In addition, the reaction of the probe with ONOO- proceeds very rapidly in aqueous solution, and the time dependence curve can reach a plateau within 1 min after the addition of four equivalences of ONOO-, whereas the probe itself is very stable, and no obvious change was observed under a continuous excitation with a laser wavelength of 370 nm (Figure S-2).

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Figure 1. Fluorescence response of probe (1 µM) to different concentrations of ONOO (0, 0.5, 1.0, 1.5, 2, 2.5, 3, 3.5, 4.0 equivalence respectively) in PBS buffer (containing 1% DMF) at 25 °C.

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Figure 2. Fluorescence response of probe (1 µM) in the presence of 10 equiv. of reactive oxygen species [1. probe, 2. . . . ONOO , 3. OCl 4. H2O2, 5. ROO , 6. NO, 7. OH, 8. O2 , 9. tBuOOH, 10. GSH (10 mM)] in PBS buffer (containing 1% DMF) at 25°C. (λex = 370 nm, λem = 501 nm, slit width: 5, 5)

Sensing Mechanism The sensing mechanism was also confirmed via LC/MS (Figure S-5).The mass spectrum shows a new peak at 291 nm, which matches the molecular weight of product 1 as depicted in Scheme 1. In addition, we obtained the product through a synthetic method that had already been confirmed via NMR and LC/MS. The peak of the UV absorbance and fluorescence emission wavelength for product 1 almost overlapped with the previous solution data (Figure S-6), indicating the formation of the desired product. One-photon Cell Imaging The utility of the probe was examined using RAW 264.7 cells for endogenous ONOOimaging. The cells were incubated with 500 ng/ml lipopolysaccharide (LPS) and 50 ng/ml gamma interferon IFN-γ for 4 h, washed with DPBS, and stained with 5 μM probe for 20 min. The probe displays more distinct and stronger fluorescence intensity (Figure 3b) than the control without any cell treatment (Figure 3a). Since ONOOis produced from nitric oxide in the cell, if the formation of nitric oxide in the cell is inhibited, the ONOO- imaging should be quenched. Accordingly, we used aminoguanidine, which is a nitric oxide synthase inhibitor, to quench the fluorescence. As expected, the fluorescence intensity was effectively quenched after pretreatment with aminoguanidine and ebselen. This result shows that our probe performs well in detecting endogenous ONOO- (Figure 3c and 3d). Moreover, a standard 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT) assay using HeLa cells was performed with different concentrations of the probe (Figure S-7). The results revealed that even after incubation with 10 μM probe for 24 h, more than 90% of the HeLa cells survived, indicating that the probe has low cytotoxicity in living cells.



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Figure 3. Fluorescence images for endogenous ONOO in RAW 264.7 cells. The cells were incubated with 500 ng/ml LPS and 50ng/ml IFN-γ for 4 h, washed with DPBS, and then stained with a 5 μM probe for 20 min. No treatment (a), LPS, IFN-γ (b), 200 μM aminoguanidine, LPS, and IFN-γ (c), and 100 μM ebselen, LPS, and IFN-γ (d). Fluorescence images were acquired via confocal microscopy. Top: ex. 405 nm/em. 490–590 nm, bottom: merged with DIC. Scale bar: 10 μM.

Two-photon Cell Imaging The successful use of the probe to detect endogenous ONOO- in live cells with onephoton microscopy led us to evaluate its utility in the two-photon mode. First, the TPA cross-sections (Φδmax) of the probe and reaction product 1 (Scheme 1) were measured using rhodamine-6G as a reference (Figure 4). As we observed in the one-photon mode, the (Φδmax) value of the probe in PBS buffer (containing 1% DMF) was negligible, whereas that of the probe with ONOO- was approximately 100 GM at 740 nm under the same conditions. This outcome suggests a sensitive turn-on response in the twophoton processes upon reaction with ONOO-. Indeed, although the two-photon microscopy image of the probelabeled RAW 264.7 cells showed a faint intensity (Figure 5a), the intensity increased markedly upon pretreatment with 500 ng/ml LPS and 50 ng/ml IFN-γ (Figure 5b and 5f). Similar results were obtained with 3morpholinosydnonimine (SIN-1), a well-established peroxynitrite donor (Figure 5c).36 The intensities decreased to a basal level upon pretreatment with aminoguanidine and ebselen (Figure 5d–e).

Figure 5. Tw0-photon excitation microscopy images of endogenous ONOO in RAW 264.7 cells. The cells were pretreated with 500 ng/ml LPS and 50 ng/ml IFN-γ for 4 h, 20 µM SIN-1for 10 min, washed with DPBS, and then stained with 5 μM probe for 20 min. No treatment (a), LPS and IFN-γ (b), SIN-1 (c), 200 μM aminoguanidine, LPS, and IFN-γ (d), and100 μMebselen, LPS, and IFN-γ (e). (f) TPEF intensity in a–e. The images were obtained by collecting emissions at 400–650 nm upon excitation at 740 nm. Scale bar: 30 µm.

Tissue Imaging We further investigated whether the probe can be used to detect ONOO- in living tissue (Figure 6). Coronal slices of the 2-day-old rat hippocampus were prepared and immediately incubated with the probe (10 μM) for 1.5 h at 37 °C. We acquired two-photon excitation microscopy (TPM) images at depths of ca. 90–180 μm to visualize the overall ONOO- distribution in the CA1 and CA3 regions (Figure 6). As observed in the cells, the tissue images showed a very weak fluorescence (Figure 6a, 6e). However, a bright signal was obtained upon pretreatment with phorbolmyristate acetate (PMA), which induced H2O2 production, and was then transformed to ONOO- by myeloperoxidase (MPO) and SIN-1 (Figure 6b, 6c).37 When we pretreated tissue with ebselen for 40 min, there was a decreased signal (Figure 6d). Moreover, we successfully observed ONOO- at the cellular level in live tissues with a higher magnification (Figure 6e–h). These results demonstrate the capability of the probe to monitor ONOO- in live tissue using TPM.

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Figure 4. Two-photon action spectra of the probe in the absence and presence of ONOO in PBS buffer (10 mM, containing 1% DMF, pH 7.4). The estimated uncertainties for the two-photon action cross-section values (Φδ) are ±15%. Figure 6.Two-photon excitation microscopy images of a rat hippocampal slices labeled with 10 μM probe for 1 h at ×10 (a-


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d) and ×100 (e-h) magnification. (a–d) Accumulated images were collected along the z-direction at a depth of approximately 90–180 μm and measured (a) before and after the addition of (b) 10 ng/ml PMA for 30min, (c) 50 μM SIN-1 for 20 min, and (d) 10 ng/ml PMA with 150 μM ebselen for 40 min. (e–h) Enlarged images of the boxed areas shown in a–d at a depth of 120 μm. (i) TPEF intensity in a–d. Images were obtained by collecting the emissions at 400–650 nm upon excitation at 750 nm. Scale bar: (a–d) 300 and (e–h) 30 μm, respectively.

CONCLUSION In summary, a new two-photon fluorescent probe for ONOO- detection was successfully designed and synthesized. The new probe exhibits excellent recognition properties, including good water solubility, with a low detection limit, low cytotoxicity, fast response, and high selectivity and sensitivity for detection of endogenous ONOOin RAW 264.7 cells. Furthermore, the probe possesses good two-photon properties with a high cross-section value (δ ≈ 100 GM), and can be used to image ONOO- in rat hippocampal slices at a depth of approximately 120 μm. Thus, the probe can be regarded as a powerful imaging tool to study peroxynitrite in living biological systems.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. 1 13 Synthetic procedures: HNMR, CNMR, MS, HRMS, Fluorescence, and UV data.

AUTHOR INFORMATION Corresponding Authors * [email protected]; fax: 82-2-3277-2385. [email protected]; fax: 82-31-219-1615.

Author Contributuibs The manuscript was written with contributions from all authors. All authors have given approval for publication of the final version of the manuscript. ‡These authors contributed equally. Notes The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS This study was supported by grants from the National Creative Research Initiative programs of the National Research Foundation of Korea (NRF) funded by the Korean government (MSIP) (No. 2012R1A3A2048814) and the National Leading Research Lab Program (No. 2016R1E1A1A02920873). Mass spectral data were obtained from the Korea Basic Science Institute (Daegu) using a Jeol JMS 700 high-resolution mass spectrometer.

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