A Far-Red-Emitting Fluorescence Probe for Sensitive and Selective

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A far red emitting fluorescence probe for sensitive and selective detection of peroxynitrite in live cells and tissues Di Wu, Jae-Chan Ryu, Youn Wook Chung, Dayoung Lee, Ji-Hwan Ryu, Joo Heon Yoon, and Juyoung Yoon Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02707 • Publication Date (Web): 18 Sep 2017 Downloaded from http://pubs.acs.org on September 18, 2017

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Analytical Chemistry

A far red emitting fluorescence probe for sensitive and selective detection of peroxynitrite in live cells and tissues Di Wu†,§, Jae-Chan Ryu‡,¶,§, Youn Wook Chung&, Dayoung Lee,† Ji-Hwan Ryu*,‡,¶, Joo-Heon Yoon*,&, #

, Juyoung Yoon*,†

†

Department of Chemistry and Nano Science, Ewha Womans University, Seoul, 120-750, Korea

‡

Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul, Korea

¶

Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea

&

The Airway Mucus Institute, Yonsei University College of Medicine, Seoul, Korea

#

Department of Otorhinolaryngology, Yonsei University College of Medicine, Seoul, Korea

§

D.W. and J.-C.R. contributed equally to this work.

*Correspondence should be addressed to: (J. Yoon) Email: [email protected], Fax: 82-2-3277-2385. (J.-H. Ryu) Email: [email protected] (J.-H. Yoon) Email: [email protected]

ABSTRACT: In this study, the far-red emitting fluorescence probe 1, containing a rhodamine derivative and a hydrazide reactive group, was developed for peroxynitrite detection and imaging. This probe, which is cell permeable and shows high sensitivity and selectivity in fluorometric detection of peroxynitrite over other ROS/RNS, was successfully utilized to detect exogenous and endogenous peroxynitrite in HeLa and RAW 264.7 cells, respectively. More importantly, 1 can also be used to detect endogenous peroxynitrite generated in Pseudomonas aeruginosa (PAO1) infected mouse bone marrow-derived neutrophils. We anticipate that the new probe will serve as a powerful molecular imaging tool in investigations of the role(s) played by peroxynitrite in a

ACS Paragon Plus Environment


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variety of physiological and pathological contexts.

INTRODUCTION Reactive oxygen (ROS) and nitrogen (RNS) species are implicated in various physiological and pathological processes.1-3 Among ROS and RNS, peroxynitrite (ONOO−) has attracted much special attention because of the key roles that it plays in signal transduction and antimicrobial activities.4-6 Peroxynitrite is endogenously produced in living systems by the diffusion-controlled coupling of nitric oxide (•NO) and superoxide radical anion (O2−•).7 Because ONOO− is more cytotoxic than •NO or O2−• alone,8 its mis-regulation in vivo is now believed to be a key contributor to numerous pathologies such as cardiovascular disease and injury, diabetes and cancer.9-12 Thus, it is of great importance to gain a better understanding of the diverse roles ONOO− plays in physiological and pathological processes.13, 14 However, owing to its short lifetime (ca. 10 ms) under physiological conditions it is not possible to directly measure ONOO− in processed cell or tissue samples by using traditional analytical methods.15-17 A promising strategy for detecting this highly reactive substance involves the introduction of fluorescence probes into cells. Accordingly, during the past several years, a number of fluorescence probes that can selectively detect ONOO− have been reported. These probes contain various reactive functional groups such as boronates,18-30 4-amino-31-33 and 4-hydroxy-benzenes,34-40 active ketones,41-43 hydrazides,44-46 selenium47-50 and tellurium species,51, 52

and others.53-66 Recently, several studies stimulated our thoughts about the use of hydrazide as

reaction group for designing probes for specific detection of ONOO−. For example, Yang and co-workers described the rhodamine B based fluorescence probe I (Scheme 1) for ONOO− using hydrazide as reaction group.44 However, interference of this probe by hypochlorite (ClO−) was not


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tested. Moreover, Zhang and co-workers recently reported the fluorescence probe II (Scheme 1), which contains dual reactive sites that can function to detect hydroxyl radicals (•OH) and hypochlorous acid (HClO) in different channel simultaneously.67 Interestingly, a hydrazide serves as the reaction group in II for detecting HClO, and its function is not interfered with by ONOO−. We reasoned that the selectivity of II might be a consequence of control offered by the fluorophore over the reactivity of the hydrazide group. If so, we envisaged that it might be possible to tune the reactivity of the hydrazide group by a proper selection of the fluorophore in which it is incorporated.68-76 This analysis led to the proposal that incorporation of the hydrazide group into rhodamine derivatives might be an ideal strategy to create probes that can selectively detect ONOO− over other ROS/RNS. It is well-known that fluorescence probes that have long absorption and emission wavelengths (far-red to NIR) possess many advantages including minimum photo-damage to biological samples, deep photon penetration in tissue and minimum interference from background auto-fluorescence of biomolecules in living systems.77-80 Despite the inspiring progress made in developing ONOO− probes, investigations of long wavelength (>600 nm) fluorescence probes with high selectivity for ONOO− are rare.13 Fortunately, a family of rhodamine type dyes with good stabilities and long absorption/emission wavelengths have been developed and successfully applied to fluorescence probes and imaging recently.81-83 Motivated by the studies described above, we hypothesized that the rhodamine derivative based fluorescence probe 1 as shown in Scheme 1, which would operate by ONOO− induced spirolactam ring opening, would serve as a far red emitting fluorescence probe for ONOO− detection. In the effort described below, we demonstrated that this probe displays a high



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sensitivity and selectivity towards peroxynitrite over other ROS/RNS and that it is cell permeable. Moreover, we utilized 1 to detect exogenous and endogenous peroxynitrite in HeLa and RAW 264.7 cells, respectively, as well as peroxynitrite generated in PAO1 infected mouse bone marrow-derived neutrophils.

Scheme 1. Structures of fluorescence probes I44, II67 and 1

EXPERIMENTAL SECTION Materials and methods. All starting reagents were obtained from commercial suppliers and used as received. GFP-tagged PAO1 was kindly provided by Sang Sun Yoon (College of Medicine, Yonsei University). 1H NMR and

13

C NMR spectra were recorded using a Bruker 300 MHz

instrument. Chemical shifts are expressed in ppm using tetramethylsilane as an internal reference, and coupling constants (J) are reported in Hz. Mass spectra were measured in the ESI mode. Fluorescence emission spectra were recorded using a FS-2 spectrophotometer (Scinco). UV absorption spectra were obtained using Evolution 201 (Thermo Scientific). All spectroscopic experiments were performed in a 1 x 1 cm quartz cell. Thin layer chromatography was conducted with 60 F254 silica plates from Merck. Silica gel 60 (0.040-0.063 mm) was used for column chromatography. Preparation of ROS/RNS. H2O2 was generated by dilution of a 28% solution in deionized water. ClO− was generated by dilution of a 5% NaClO solution in deionized water. Hydroxyl radical (∙OH) was generated by using the Fenton reaction. For this purpose, hydrogen peroxide (H2O2,


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100 μM) was added to a solution of ammonium iron (II) sulfate (100 μM) in deionized water. Superoxide solution (∙O2−) was prepared by adding KO2 to dry dimethylsulfoxide. Singlet oxygen (1O2) was generated in situ by addition of a H2O2 stock to a solution containing 10 equivalents of HClO. NO2− and NO3− were prepared by adding NaNO2 and NaNO3, respectively, to deionized water. ROO∙ was prepared by adding 2,2'-azobis-(2-amidinopropane) dihydrochloride to deionized water. Tert-butyl hydroperoxide was generated by dilution of the commercial aqueous solutions (70%) with deionized water. NO∙ was generated from SNP (sodium nitroferricyanide (III) dihydrate). ONOO− was prepared using the previously described procedure and the concentration of peroxynitrite was estimated by using extinction co-efficient of 1670 cm−1M−1 at 302 nm in 0.1 M sodium hydroxide aqueous solutions.84 Cell Culture and Confocal Microscopy. The HeLa cells and RAW 264.7 cells (mouse macrophage cells) were cultured on the surface of a glass slide in Dulbecco’s modified Eagle medium SPP medium at 37 °C in 5% CO2. For cell imaging experiments, the Hela cells were incubated with 1 (10 μM) for 30 min, washed with DPBS buffer,85 and then treated with SIN-1 (50 μM) for 30 min. For a blocking experiment, cells were pre-cultured with ebselen for 30 min. Images were obtained by collecting the emissions at 575−675 nm upon excitaƟon at 559 nm using a confocal imaging system. For the detection of endogenous ONOO−, RAW 264.7 cells were treated with bacterial endotoxin lipopolysaccharide (LPS) (1 μg/ml) for 4 h, IFN-γ (50 ng/mL) for 1 h and co-incubated with 1 (10 μM) for 30 min. For a blocking experiment, cells were pre-cultured with ebselen for 30 min before being incubated with 1. Fluorescence images were recorded using a confocal imaging system as mentioned above. The experimental details for detection of immune associated ONOO− in mouse bone marrow-derived neutrophils are given in supporting



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information. Synthesis of 1. Intermediates 2 and 3 in the pathway for synthesis of the rhodamine based probe 1 were prepared utilizing literature procedures.81 A mixture of hydrazine hydrate (50 mg, 1.0 mmol) and PyBOP (104 mg, 0.2 mmol) were added to a solution of 3 (113 mg, 0.2 mmol) in dry dichloromethane. The mixture was vigorously stirred at room temperature for 3 h and then concentrated in vacuo. The residue was subjected to silica gel chromatography to afford 1 as a white solid (82 mg, 85%). 1H NMR (300 MHz, CDCl3) 7.97– 7.90 (m, 1H), 7.73 (d, J = 8.6, 1H), 7.51–7.42 (m, 2H), 7.23– 7.20 (m, 1H), 6.66–6.63 (m, 1H), 6.50–6.46 (m, 2H), 6.38 (d, J = 8.8, 1H), 6.32–6.28 (m, 1H), 3.67 (s, 2H), 3.35 (q, J = 7.0, 4H), 2.98 (s, 6H), 2.70–2.62 (m, 2H), 1.89–1.79 (m, 1H), 1.72–1.61 (m, 1H), 1.17 (t, J = 7.0, 6H). 13C NMR (CDCl3, 75 MHz)

166.2, 153.4, 150.6, 149.5, 148.6, 147.7, 137.9, 132.3, 130.8,

128.3,127.9, 123.5, 123.1, 122.9, 118.3, 111.2, 109.7, 108.4, 104.0, 99.4, 98.0, 67.4, 44.3, 40.4, 28.8, 21.2, 12.6. HRMS (ESI) calcd. for C30H33N4O2+ [M+H+] 481.2598, found 481.2610.

RESULTS AND DISCUSSION Probe design and synthesis. In order to explore the proposal that it would serve as a fluorescence probe for ONOO−, the rhodamine based hydrazide 1 was synthesized using the three steps route shown in Scheme 2. By using procedures described in the literature,81 commercially available 6-amino-1-tetralone was first converted to its N,N-dimethyl derivative 2, which was then transformed to 3. Condensation of 3 with hydrazine monohydrate in the presence of (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP) then produced 1 in 85% yield (Scheme 2).


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Scheme 2. Route for preparation of 1

Sensitivity and proposed mechanism for sensing of ONOO− by 1. The capability of using 1 to track ONOO− in buffer solution (PBS, 1.0 mM, PH = 7.4) was explored first. As shown in Figure 1, the free probe 1 is colorless and almost non-fluorescent in PBS buffer as an expected consequence of the presence of the spirocyclic ring system which prevents conjugation of the two amine-substituted arene rings. Addition of ONOO− to a PBS solution of 1 (10 μM, pH 7.4) results in dose dependent appearance of an absorption band centered at 600 nm in association with development of a pale blue color (Figure S1). This change is caused by spirolactam ring opening and the resulting generation of a rhodamine like chromophore (Figure S1). Moreover, addition of ONOO− results in dramatic, rapid and dose-dependent development of fluorescence band with a maximum at 638 nm (Figure 1A). The fluorescence intensity increase at 638 nm reaches a plateau when the amount of ONOO− is >40 μM (Figure S2), which corresponds to a fluorescence enhancement of ~80 folds. Furthermore, good linearity was observed between the fluorescence enhancement at 638 nm and the concentration of ONOO− in the range of 0–34 μM (Figure 1B). The detection limit of 1 was estimated to be 45 nM based on the formula of 3σ/k, which indicates that this probe



is well suited for highly sensitive ONOO− detection. The mechanism of the ONOO− promoted reaction that causes the color change and fluorescence enhancement of related spirolactams has been demonstrated in previous studies.14 Thus, only a simple experiment was performed to show that this pathway is followed in the process taking place between 1 and ONOO− (Figure S3). Specifically, mass spectrometric (MS) analysis of the mixture formed by reaction of 1 with ONOO− shows that a new peak at 467.2345 (m/z) arises, which is well-matched with the calculated value (467.2329 (m/z)) of the proposed product (Figure S3).

(B) 75 Fold of fluorescence enhancement

(A) 1000

800 Fluorescence intensity

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100 µM 600

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Figure 1. (A) Fluorescence response of 1 (10 μM, PBS buffer, pH 7.4) to various concentrations of upon addition of ONOO− (0–100 μM); (B) Linear correlation between the enhancement of emission intensity at 638 nm and the concentration of ONOO− (0–34 μM). Spectra were collected after incubation with different concentrations of ONOO− for 3 min. λex = 600 nm, λem = 638 nm. Slits: 5/5 nm.

Selectivity of 1 towards ONOO−. The response of 1 towards a panel of ROS and RNS was tested to establish its selectivity for detecting ONOO− (Figure 2A). The results show that, the fluorescence enhancement of 1 only can be triggered by ONOO− and there are no obvious changes in its fluorescence when other ROS/RNS are added (Figure 2B). To further evaluate the potential of applying 1 for ONOO− detection in biochemical systems, the effect of pH on the


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fluorescence response of 1 to ONOO− was investigated. As shown in Figure S4, there is no significant change in the fluorescence turn-on response to ONOO− of 1 at 638 nm over the pH 5-11 range and that the response to ONOO− is constant within the biologically relevant pH range from 6 to 9. This broad pH tolerance indicates that 1 should have potential applications in detection of ONOO− under physiological condition.

(B) 100 Fold of fluorescence enhancement

(A) 1000

800 Fluorescence intensity

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-

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400 1 and 1+other ROS/RNS 200

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1

1. Ref, 2. H2O2, 3. ClO , 4. •OH, 5.•O2 , 6. O2, 7. NO2 , t 8.NO3 , 9. ROO•, 10. BuOO•, 11.NO•, 12.ONOO

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Figure 2. Fluorescence response (A) and relative fluorescence intensities (B) of 1 (10 μM, PBS buffer, pH 7.4) in the absence and presence of ONOO− (50 μM) and other ROS/RNS (100 μM).

Imaging exogenous ONOO− in HeLa Cells. Encouraged by the results described above, we next examined the potential use of 1 to visualize ONOO− in live cells by using confocal fluorescence microscopy. We observed that HeLa cells incubated with 1 (10 μM) for 30 min display faint fluorescence (Figure 3a). In contrast, cells pre-incubated with 1 (10 μM) gave bright red fluorescence after treatment with the ONOO− generator 3-morpholinosydnonimine hydrochloride (SIN-1) (Figure 3b). Moreover, the red fluorescence is significantly reduced by the pre-treatment with the ONOO− scavenger, ebselen (Figure 3c). These observations indicate that 1 is a promising probe for detection of exogenous ONOO− in live cells.



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Figure 3. Fluorescence in live cells. HeLa cells were incubated with 1 (10 μM) for 30 min and washed with DPBS. To the cells were then added (a) 0 and (b) 50 μM SIN-1. (c) HeLa cells were incubated with 1 (10 μM) for 30 min, pre-treated with 50 μM ebselen, and then treated with 50 μM SIN-1 for 30 min. Fluorescence images were collected on confocal microscopy. λex 559 nm/λem 575–675 nm.

Imaging endogenous ONOO− in RAW 264.7 cells. RAW 264.7 macrophages, which are known to generate ROS/RNS in inflammatory and immunological processes, were employed as a bioassay model to further demonstrate the utility of 1 for imaging endogenous ONOO−. Cellular endogenous ONOO− was generated by incubating RAW 264.7 cells with LPS (1 µg/mL) for 4 h and interferon-γ (IFN-γ, 50 ng/mL) for 1 h. As can be seen by viewing Figure 4, cells that are not pretreated with LPS and IFN-γ display faint fluorescence signals after incubation with 1 (10 μM) for 30 min. In sharp contrast, cells stimulated with LPS and IFN-γ gave bright red fluorescence signals after incubation with 1. To gain further information, an experiment was carried out utilizing ebselen to scavenge ONOO− produced by the stimulation of RAW 264.7 cells by treatment with LPS and IFN-γ. As expected, the bright cellular fluorescence seen in stimulated cells is heavily attenuated in those pretreated with ebselen. The combined results indicate that 1 can be utilized to image endogenous ONOO− in RAW 264.7 cells and, more significantly, that it is effective small molecule fluorescence probe for detecting ONOO− in biological systems.


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Figure 4. Detection of peroxynitrite produced by an immune reaction in the macrophage. RAW 264.7 cells were treated with (a) 0 and (b) 1 μg mL-1 LPS 4 h, 50 ng mL-1 IFN-γ 1 h and (c), LPS, IFN-γ + 50 μM ebselen. The cells were incubated with 1 (10 μM) for 30 min and washed with DPBS and then imaged by confocal microscopy. λex 559 nm/λem 575–675 nm.

Imaging endogenous ONOO− in PAO1 infected mouse bone marrow-derived neutrophils. ONOO− is known to be produced in neutrophils upon treatment with bacterial motifs.86 As a result, we further explored the applicability of 1 by employing it to detect ONOO− in PAO1 infected mouse bone marrow-derived neutrophils (BMDNs).87 First, we examined whether 1 could be employed to detect ONOO− generated in BMDNs that are treated with the ONOO− donor SIN-1. The result shows that the fluorescence intensity of 1 pretreated with BMDNs increases in a time-dependent manner following addition of SIN1, whereas no obvious changes in fluorescence from the BMDNs occurs in the absence of SIN1 (Figure S5a). It has been reported that Nox2 is responsible for ONOO− generation in phorbol myristate acetate (PMA)-stimulated neutrophil-like cells.88 Therefore, we investigated whether 1 could be utilized to detect changes in ONOO− concentrations in BMDNs extracted from wild type mice (Nox2+/+) or Nox2-deficient mice (Nox2-/-) infected ex vivo (i.e., isolated primary neutrophils which were infected with pathogen such as PAO1). Inspection of the images displayed in Figure S5b demonstrates that fluorescence



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of 1 is more intense in PAO1-infected Nox2+/+ BMDNs. However, no obvious changes in the fluorescence intensity of 1 take place in PAO1-infected Nox2-/- BMDNs or PAO1-infected Nox2+/+ BMDNs that are pre-treated with NOX inhibitor diphenyleneiodonium (DPI). Confocal microscopy was also employed to demonstrate that 1 can be applied to monitor increases in the concentrations of ONOO− in Nox2+/+ BMDNs infected by GFP-tagged PAO1. This method was also used to show that ONOO− production does not occur in Nox2-/- BMDNs by GFP-tagged PAO1 (Figure 5). Additionally, the results of an experiment using a lung infection mouse model indicates that 1 can utilized to detect an increase of ONOO− in neutrophils from BAL fluids in PAO1-infeced Nox2+/+ mice, and that ONOO− is not produced in PAO1-infected Nox2-/- mice (Figure 6). The results outlined above suggest that 1 may be applicable to monitoring respiratory infectious diseases through specific detection of ONOO− in neutrophils ex vivo and in vivo. In mammalian systems, uncontrolled ONOO− generation in innate immune cells, including neutrophils and macrophages, may lead to ailments such as inflammatory bowel and neurodegerative diseases.6 Therefore, ONOO− detection by using 1 could play an important role in the diagnosis of these ailments. Additionally, 1 may serve as a promising tool for evaluating the abilities of therapeutic candidates for the inhibition of unwarranted ONOO− generation in neutrophils.


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Figure 5. Confocal microscope images of GFP-tagged PAO1 and 1 in BMDNs. Images of neutrophils derived from Nox2

+/+

(top) or Nox2

−/−

(bottom) mice treated with GFP-tagged PAO1 and 1 for 1 h. Images of GFP-tagged PAO1

(green) were obtained were obtained by collecting the emissions at 500−550 nm upon excitaƟon at 488 nm. Images of 1 (red) were obtained by collecting the emissions at 630−660 nm upon excitaƟon at 561 nm. Scale bars, 20 μm.

Figure 6. Flow cytometry analysis of 1 in neutrophils derived from BAL fluids in a lung infected mouse model. Results of Ly-6G-positive neutrophils derived from Nox2+/+ or Nox2−/− mice after being treated with 1 × 105 CFU of PAO1 for 7 h. FSC, forward-scattered light; SSC, side-scattered light.



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CONCLUSION In summary, in this study we developed a new, rhodamine derivative based fluorescence probe for detecting ONOO− that functions by reaction of a hydrazide group. The probe displays high sensitivity and selectivity to ONOO− over other ROS/RNS. Furthermore, the product of reaction of 1 and ONOO− displays long wavelength absorption (600 nm) and emission (638 nm) bands. Additionally, the probe was employed to image exogenous ONOO− in HeLa cells and endogenous ONOO− in RAW 264.7 cells as well as in neutrophils of a mouse infection model. Collectively, the observations made in this investigation show that the new probe possesses high potential applications in uncovering the physiological and pathological roles of ONOO− in living systems.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This research 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), Global Research Laboratory (GRL) Program of the National Research Foundation (NRF) funded by Ministry of Science, ICT, Future Planning (2016K1A1A2910779) and the Bio & Medical Technology Development Program of the

NRF

funded

by

the

Ministry

of

Science,

ICT

&

Future

Planning

(2016M3A9D5A01952415).

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website


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Synthetic procedures, cell imaging procedures, 1H NMR and

13C

NMR analyses, HRMS,

fluorescence, and UV data.

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