Subscriber access provided by CORNELL UNIVERSITY LIBRARY
Article
Rational Design of an #-Ketoamide-Based Near-Infrared Fluorescent Probe Specific for Hydrogen Peroxide in Living Systems Xilei Xie, Xiu\'e Yang, Tianhong Wu, Yong Li, Mengmeng Li, Qi Tan, Xu Wang, and Bo Tang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b01256 • Publication Date (Web): 21 Jul 2016 Downloaded from http://pubs.acs.org on July 22, 2016
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 7
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry
Rational Design of an α-Ketoamide-Based Near-Infrared Fluorescent Probe Specific for Hydrogen Peroxide in Living Systems Xilei Xie,a Xiu’e Yang,a Tianhong Wu,a Yong Li,a Mengmeng Li,a Qi Tan,b Xu Wang,* a and Bo Tang* a a
College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong Normal University, Jinan 250014, P. R. China b Shandong Provincial Hospital affiliated to Shandong University ABSTRACT: Hydrogen peroxide, an important biomolecule, always receives the earnest attention because of its physiological and pathological functions. In this paper, we present the rational design, characterization, and biological application of a mitochondriatargetable NIR fluorescent sensor, Mito-NIRHP, for hydrogen peroxide visualization. Mito-NIRHP utilizes a unique reaction switch, α-ketoamide moiety, to turn on a highly specific, sensitive, and rapid fluorescence response toward hydrogen peroxide coupled with the intramolecular charge transfer strategy. Mito-NIRHP is competent to track endogenously produced hydrogen peroxide in both living cells and living animals. In addition, utilizing Mito-NIRHP, overgeneration of hydrogen peroxide during ischaemia-reperfusion injury was directly visualized at both cell and organ levels.
Hydrogen peroxide (H2O2) is a main member of the reactive oxygen species (ROS) family and is involved in a wide range of physiological and pathological processes.1,2 To date, H2O2 has been acknowledged as an oxidative stress marker in aging and disease,2 a defense agent in immune response to pathogen invasion,3 and a messenger molecule related to cell proliferation, migration, and differentiation.4 There is increasing convincing evidence that aberrant production of H2O2 contributes to various pathological processes, such as diabetes,5 cancers,6 cardiovascular disorders,7 and neurodegeneration,8 etc. To achieve the complete elucidation of H2O2 bio-function, an analytical tool capable of monitoring its generation, distribution, and dynamic fluctuation in living systems is desirable. Fluorescence imaging based on small-molecule fluorescent probes have been emerged as an efficient methodology to visualize the spatial and temporal distribution of biomolecules in biological specimens due to its real-time, sensitive, and noninvasive characteristics.9–12 Compared with other ROS, H2O2 is a relatively stable species and exhibits mild reactivity. Therefore, fluorescent probes for H2O2 are liable to be interfered by highly reactive species, such as peroxynitrite.13 Up to date, numerous fluorescent sensors for H2O2 have been constructed based on different chemical strategies, including H2O2 mediated hydrolysis of sulfonic esters,14,15 H2O2 triggered conversion of arylboronates to phenols,16–22 transformation from benzil to benzoic acid,23 and so on.24–26 However, the probes based on sulfonic esters hydrolysis also respond to other ROS27 and thiols28 besides H2O2.12 And it is reported that several boronate-based probes react more slowly with H2O2 as compared to other reactive species (e.g., peroxynitrite and hypochlorite).13,29,30 Some of them have been used to detect peroxynitrite31–33 or benzoyl peroxide34 instead of H2O2. Therefore, there still has been a large demand to develop new fluorescent
probe which features high specificity, excellent sensitivity and fast response toward H2O2. In this paper, we identified the α-ketoamide moiety as a novel reaction switch specific for H2O2. Integration of this unique moiety into the hemicyanine fluorophore afforded a fluorescence “off-on” probe, Mito-NIRHP, for highly specific and sensitive detection of H2O2, which features NIR absorption/emission and mitochondrial localization. The practical utility of Mito-NIRHP in biological contexts was demonstrated by imaging basal and endogenous H2O2 levels in living cells as well as in living animals. Moreover, Mito-NIRHP was utilized as an efficient molecular tool to prove that H2O2 was overgenerated during ischaemia-reperfusion injury (IRI) at both cell and organ levels, corroborating the close relationship between H2O2 and this type of organism damage process.
EXPERIMENTAL SECTION Materials and instruments. Unless otherwise stated, all reagents for synthesis were purchased from commercial suppliers and were used without further purification. The solvents were purified by conventional methods before use. All synthesis was carried out under an argon atmosphere, magnetically stirred and monitored by thin-layer chromatography (TLC). Flash chromatography was performed using the silica gel (100–200 mesh). 3-(4,5-Dimethyl-thiazol-2-yl)-2,5diphenyltetrazoliumbromide (MTT) and phorbol myristate acetate (PMA) were purchased from Sigma Chemical Company. Rotenone was from Bide Pharmatech. Ltd. (Shanghai, China). Mito-Tracker Green was purchased from Thermo Fisher Scientific (Shanghai, China). The MGC AnaeroPouch®, oxygen indicator, and anaerobic bag were purchased from Mitsubishi Gas Chemical Co. (Tokyo, Japan). All the cells were purchased from Cell Bank of the Chinese Academy
ACS Paragon Plus Environment
Analytical Chemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
of Sciences (Shanghai, China). Male Kunming mice (20 g) were purchased from School of Medicine at Shandong University. All animal experiments were in agreement with the guidelines of the Institutional Animal Care and Use Committee. Sartorius ultrapure water (18.2 MΩ. cm) was used throughout the experiments. Fluorescence spectra were obtained by a FLS-980 Edinburgh Fluorescence Spectrometer (Edinburgh Instruments Ltd, England) with a Xenon lamp and 1.0-cm quartz cells at the slits of 2.0/2.0 nm. Absorption spectra were recorded on a UV-1700 spectrophotometer (Shimadzu, Japan). pH measurements were performed with a pH-3c digital pH-meter (Shanghai Lei Ci Device Works, Shanghai, China). 1H NMR and 13C NMR spectra were taken on a Bruker Advance 300MHz spectrometer and a 400MHz spectrometer (Bruker Co., Ltd., Germany), δ values are in ppm relative to TMS. HRMS analysis was carried out on a Bruker maxis UHR-TOF Ultra High Resolution Quadrupole-time of flight mass spectrometer (Bruker Co., Ltd., Germany). The fluorescence images of cells were taken using a LSM 880 confocal laser scanning microscopy (Zeiss Co., Ltd. Germany) with an objective lens (×20). In vivo imaging was performed on IVIS Lumina III system (Xenogen, USA) with a metal halide lamp (150 W). The absorbance was measured using a TRITURUS microplate reader in the MTT assay. Synthesis of Mito-NIRHP. Cy-NH2 was synthesized according to reported procedure.35 A mixture of 2-(4nitrophenyl)-2-oxoacetic acid (39 mg, 0.2 mmol), oxalyl chloride (53 µL, 0.6 mmol), DMF (2 drops), and dichloromethane (3 mL) was refluxed for 1 h and then evaporated. To this crude product, 5 mL dichloromethane was added, and then Cy-NH2 (52 mg, 0.1 mmol) and triethylamine (12 µL, 0.4 mmol) were added. The reaction mixture was stirred at room temperature for 30 min, poured into saturated NaHCO3 solution (50 mL), and extracted with 50 mL dichloromethane three times. Combination of the extraction, the organic solvent was dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The residue was purified by column chromatography on silica gel (dichloromethane/ethanol, 50:7 v/v) to afford a blue solid (65 mg, 93% yield). 1H NMR (300 MHz, DMSO-d6) δ 11.57 (s, 1H), 8.60 (d, J = 15.1 Hz, 1H), 8.42 (d, J = 9.0 Hz, 2H), 8.33 (d, J = 9.0 Hz, 2H), 8.07 (s, 1H), 7.81 (d, J = 7.2 Hz, 1H), 7.73 (d, J = 8.3 Hz, 2H), 7.61 (d, J = 8.5 Hz, 1H), 7.56 (td, J = 7.9, 1.0 Hz, 1H), 7.51–7.43 (m, 2H), 6.65 (d, J = 15.1 Hz, 1H), 4.48 (q, J = 6.9 Hz, 2H), 2.86–2.60 (m, 4H), 1.90– 1.83 (m, 2H), 1.76 (s, 6H), 1.39 (t, J = 7.1 Hz, 3H); 13C NMR (75 MHz, DMSO-d6) δ 186.93, 177.53, 161.70, 159.65, 152.64, 150.50, 145.28, 142.28, 140.98, 140.58, 137.61, 131.74, 129.65, 128.97, 128.24, 127.43, 123.84, 122.96, 118.36, 117.36, 114.17, 113.37, 106.48, 105.23, 99.55, 62.99, 50.62, 27.18, 23.01, 19.85, 14.02, 12.81; HRMS (ESI): calcd for C35H32N3O5 (M+), 574.2336; found, 574.2365. Synthesis of Compound 1. This analog was synthesized according to the above procedure starting from pyruvic acid instead of 2-(4-nitrophenyl)-2-oxoacetic acid. 1H NMR (400 MHz, DMSO-d6) δ 10.93 (s, 1H), 8.56 (d, J = 16 Hz, 1H), 8.09 (s, 1H), 7.84 (d, J = 8 Hz, 1H), 7.78 (d, J = 8 Hz, 1H), 7.74 (d, J = 8 Hz, 1H), 7.55 (s, 1H), 7.53 (s, 1H), 7.49–7.46 (m, 1H), 7.42 (s, 1H), 6.62 (d, J = 16 Hz, 1H), 4.47 (s, 2H), 2.71 (s, 2H), 2.67 (s, 2H), 2.45 (s, 3H), 1.81 (s, 2H), 1.76 (s, 6H), 1.38 (s, 3H); HRMS (ESI): calcd for C30H31N2O3 (M+), 467.2329; found, 467.2318.
Page 2 of 7
Synthesis of Compound 2. This analog was synthesized according to the above procedure starting from 2-oxo-2phenylacetic acid instead of 2-(4-nitrophenyl)-2-oxoacetic acid. 1H NMR (400 MHz, DMSO-d6) δ 11.50 (s, 1H), 8.61 (d, J = 16.0 Hz, 1H), 8.08 (d, J = 8Hz, 2H), 8.05 (s, 1H), 7.82– 7.77 (m, 2H), 7.73 (d, J = 8 Hz, 1H), 7.66–7.59 (m, 4H), 7.55 (t, J = 8 Hz, 1H), 7.47 (s, 2H), 6.65 (d, J = 16 Hz, 1H), 4.48 (d, J = 4 Hz, 2H), 2.75 (s, 2H), 2.70 (s, 2H), 1.85 (s, 2H), 1.76 (s, 6H), 1.39 (s, 3H); HRMS (ESI): calcd for C35H33N2O3 (M+), 529.2485; found, 529.2405. Cell confocal imaging experiment. HepG2 cells were seeded at a density of 1 × 106 cells mL-1 in high-glucose DMEM supplemented with 10% fetal bovine serum, 1% penicillin, and 1% streptomycin. Cultures were maintained in a humidified incubator MCO-15AC (SANYO, Japan) at 37 °C, in 5% CO2/95% air. Cells were passed and plated on 18 mm glass coverslips in culture dish. The cells were excited at 633 nm with a laser and the emission was collected between 660–750 nm. Animal models and in vivo imaging. Before imaging, the Kunming mice were fasted for 12 h to avoid the possible food fluorescence interference to the dye fluorescence. The mice were divided into three groups. The first group was given an injection of Mito-NIRHP (20 µM, 100 µL) into the peritoneal cavity as the negative control. The second group was given an injection of rotenone (2.5 mM, 100 µL, thus 5 mg/kg of animal weight) into the peritoneal cavity and followed by injection of Mito-NIRHP (20 µM, 100 µL) at the same region after 1 h. The third group was successively treated with rotenone (2.5 mM, 100 µL) for 1 h, NAC (20 mM, 100 µL, thus 16 mg/kg of animal weight) for 1 h, and Mito-NIRHP (20 µM, 100 µL) at the same region. Before imaging, the mice were anesthetized with 4% chloral hydrate (15 mL/kg) by intraperitoneal injection. Then the whole body images of the mice were acquired using an IVIS Lumina III system with 660 nm excitation and 710 nm emission channel. Confocal imaging during oxygen-glucose deprivation/reperfusion (OGD/R). HepG2 cells were washed twice with PBS and then cultured with DMEM without glucose that had been pre-gassed with 95% N2-5% CO2 for 10 min to remove residual oxygen. The dishes were placed into an airtight AnaeroPouch® bag, which provided near anaerobic conditions with an O2 concentration
