Accepted Manuscript A novel mitochondria-targeted two-photon fluorescent probe for dynamic and reversible detection of the redox cycles between peroxynitrite and glutathione Chunlong Sun, Wen Du, Peng Wang, Yang Wu, Baoqin Wang, Jun Wang, Wenjun Xie PII: S0006-291X(17)32101-0 DOI: 10.1016/j.bbrc.2017.10.123 Reference: YBBRC 38740 To appear in: Biochemical and Biophysical Research Communications Received Date: 22 October 2017 Accepted Date: 23 October 2017 Please cite this article as: C. Sun, W. Du, P. Wang, Y. Wu, B. Wang, J. Wang, W. Xie, A novel mitochondria-targeted two-photon fluorescent probe for dynamic and reversible detection of the redox cycles between peroxynitrite and glutathione, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.10.123. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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. AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 1 A novel mitochondria-targeted two-photon fluorescent probe for 2 dynamic and reversible detection of the redox cycles between 3 peroxynitrite and glutathione RI PT 4 5 Chunlong Suna, 1∗, Wen Dua, 1∗∗, Peng Wangb, Yang Wuc, Baoqin Wanga, Jun Wanga, 6 Wenjun Xiea 7 a. School of biotechnology, Key Laboratory of Instrumental Analysis of Binzhou City, Shandong 9 Provincial Key Laboratory of Eco-environmental Science for Yellow River Delta, Binzhou SC 8 University, Binzhou 256603, China 11 b.Department of Biomedical Engineering, School of Engineering, China Pharmaceutical University, 12 Nanjing, 210009, China 13 c. Research Center of Clinical Oncology, Jiangsu Cancer Hospital, Nanjing 210009, China 15 TE D 14 M AN U 10 Abbreviations: ONOO−, peroxynitrite; GSH, glutathione; GSSG, glutathione 17 disulfide; ROS reactive oxygen species; D-π-A, donor-π-acceptor; OP, One-photon; 18 TP, Two-photon; MTT, 3-(4,5-Dimethythiazol-2-yl)-2,5-diphenyl tetrazolium 19 bromide; L929, Mouse fibroblast cell line; MCF-7, human breast adenocarcinoma cell 20 line; Colo 205, human colon adenocarcinoma cells; PET, photoinduced electron 21 transfer; SIN-1, 3-morpholinosydnonimine AC C 22 EP 16 23 *Corresponding author. Tel.: +86-0543-3190096; E-mail address: email@example.com (Chunlong Sun) ** Corresponding author. Tel.: +86-0543-3190096; E-mail address:firstname.lastname@example.org (Wen Du) 1 These authors contribute equally. 1 ACCEPTED MANUSCRIPT Abstract 25 Redox homeostasis is important for maintenance of normal physiological functions 26 within cells. Redox state of cells is primarily a consequence of precise balance between 27 levels of reducing equivalents and reactive oxygen species. Redox homeostasis 28 between peroxynitrite (ONOO−) and glutathione (GSH) is closely associated with 29 physiological and pathological processes, such as prolonged relaxation in vascular 30 tissues and smooth muscle preparations, attenuation of hepatic necrosis, and activation 31 of matrix metalloproteinase-2. We report a two-photon fluorescent probe (TP-Se) 32 based on water-soluble carbazole-based compound, which integrates with organic 33 selenium, to monitor changes in ONOO−/GSH levels in cells. This probe can reversibly 34 respond to ONOO− and GSH and exhibits high selectivity, sensitivity, and 35 mitochondrial targeting. The probe was successfully applied to visualize changes in 36 redox cycles during ONOO− outbreak and antioxidant GSH repair in cells. The probe 37 will lead to significant development on redox events involved in cellular redox 38 regulation. 39 Key Words 40 Redox; Two-photon fluorescent probe; Mitochondria; TP-Se; Fluorescence imaging 43 44 45 46 47 SC M AN U TE D EP 42 AC C 41 RI PT 24 48 49 50 51 52 2 ACCEPTED MANUSCRIPT 53 1. Introduction Mitochondria, the principal energy-producing compartments in most cells, play roles 55 in numerous vital cellular processes . Endogenous peroxynitrite (ONOO−) has been 56 recognized as a strong oxidant agent in cells, especially in mitochondria. In vivo, high 57 concentrations of ONOO− are produced from the fast reaction with nitric oxide (NO) 58 and superoxide anion (O2•−), and this reaction doesn't have to be enzymatic. The 59 ONOO− is relatively stable, but the acid form (ONOOH) rapidly become to nitrate. 60 Although the half-life of the acid form (ONOOH) is very short, the oxidative species 61 comes into being a unique biological oxidation_reduction cycle indicating human 62 health and disease [2, 3]. ONOO− has also been shown as a nitrating agent that causes 63 nitrative stress in cells, but more investigations concerning endogenous ONOO− have 64 been devoted to modulating signal transduction pathways via its ability to form nitrate 65 biomolecules, including nitrated tyrosine residues, 8-nitroguanosine, and nitro fatty 66 acids, to influence cellular processes[4-7]. Intracellular thiols provide abundant 67 reducing sources, which are central to cellular redox homeostasis in antioxidant defense 68 systems. Glutathione (GSH) is the most abundant endogenous thiol, whose 69 concentration ranges from 1 mM to 15 mM depending on cell type [8-11]. GSH 70 controls redox homeostasis through equilibrium between its reduced (GSH) and 71 disulfide forms (GSH disulfide (GSSG)). Formation and reactions of intracellular 72 peroxynitrite primarily occur mitochondria [12-15], wherein GSH exists predominantly 73 in reduced form at a GSH/GSSG molar ratio of >100:1[16, 17]. ONOO− can 74 significantly perturb mitochondrial GSH/GSSG ratio and cause irreversible damage to 75 respiration. Interpreting regulation and interplay between peroxynitrite and Glutathione 76 can also reveal physiological and pathological roles of endogenous peroxynitrite. 77 AC C EP TE D M AN U SC RI PT 54 ONOO− is modulated by cellular antioxidant defense systems, and the selenium (Se) 78 plays an important role as an active target of the antioxidant enzyme glutathione 79 peroxidase (GPx) [18, 19]. GPx can catalyze the reduction of ONOO− by GSH via a 80 particular ping-pong mechanism . Small artificial organic molecule probes 81 emerged as one of the most powerful biotechniques for detecting physiologically active 3 ACCEPTED MANUSCRIPT species in living systems with high temporal and spatial resolution . Not only are 83 these probes confirmed useful in detection of disease states, but they also allow for 84 screening-type analysis of potential signal transduction pathways in cells. Therefore, 85 we strive to develop a probe that can respond reversibly to changes in redox 86 homeostasis regulation via a redox-based mechanism for visualizing states of these 87 redox cycles. RI PT 82 One-photon (OP) fluorescence imaging offers a powerful approach to monitor 89 intracellular ONOO− levels owing to its high sensitivity . However, existing 90 ONOO− fluorescent probes used for cell imaging feature some drawbacks. For instance, 91 small molecular fluorescent probes frequently suffer from interference from other 92 reactive oxygen species (ROS) , delayed response time, and irreversible reactions 93 . Two-photon (TP) imaging possesses distinct advantages, including increased 94 penetration depth and reduced specimen photodamage, due to its excitation that utilizes 95 two low-energy photons . Although several TP probes have been used for imaging 96 thiols , H2O2 , and metal ions , no report exists on TP imaging of ONOO− in 97 live cells. For ideal monitoring of ONOO− fluxes, fluorescent probes must offer more 98 substantial penetration depth, less photodamage, and high sensitivity, instantaneous 99 response, and reversibility considering the extremely short lifetime and low 101 M AN U TE D concentrations of intracellular ONOO−. EP 100 SC 88 Therefore, a novel probe with features of TP fluorescence imaging should be constructed for dynamically monitoring fluctuations of redox selectively, 103 instantaneously, and reversibly. This imaging method not only can considerably 104 improve accuracy of ONOO− detection but can also be adopted flexibly according to 105 depths of various samples. To date, no reports have been published regarding the use of 106 TP fluorescent probes in imaging redox, especially in mitochondria. After confirming 107 the key point of our research, we designed and synthesized a single fluorescent TP-Se 108 probe for reversible detection of ONOO− and GSH (Scheme 1) [29-31]. AC C 102 4 ACCEPTED MANUSCRIPT O Se Se N N I I ONOO N N GSH N 109 N TP-Se TP-SeO Scheme 1. Structures of TP-Se and TP-SeO. RI PT 110 A novel TP fluorophore was utilized based on electron-donating carbazole and 112 electron-withdrawing cationic pyridinium moiety. This excellent intermolecular charge 113 transfer system will guarantee TP activity of the probe [32-35]. As for TP-Se, methyl 114 pyridinium moiety as mitochondria-targeted functional group was conjugated with 115 carbazole to identify location of mitochondria [36, 37] and also provided the probe with 116 good water solubility. Considering these advantages, we mimicked the catalytic cycle 117 and developed a TP probe containing an organoselenium moiety that can be used for 118 reversible peroxynitrite detection. 119 2. Experimental 120 2.1. Apparatus and Materials M AN U SC 111 Two-photon excited fluorescence spectra were measured using a Tsunami 3941: Ti: 122 sapphire femtosecond laser as exciting light source (865 nm) with a pulse width of 123 <145 fs and a repetition rate of 75 MHz, and USB2000 (OCEAN OPTICS) was used as 124 the recorder. A UV-Vis Spectrophotometer (SHIMADZU, Japan) was used to perform 125 the absorbance measurements. A Nikon confocal laser-scanning microscope (NIKON, 126 Japan) with an objective lens was used to perform the florescence measurements. 127 Compounds 2 and 3 were synthesized in our laboratory. 128 2.2. Design and Synthesis of two-photon fluorescent probe TP-Se AC C EP TE D 121 129 130 131 Scheme 2. Synthetic route of two-photon fluorescent probe TP-Se. Scheme 2 shows the synthetic route of designed two-photon fluorescent probe 5 ACCEPTED MANUSCRIPT TP-Se. Compound 2 (0.6 mmol) and Compound 3 (1.80 mmol) were dissolved in 133 Methanol (150 ml). The solution was stirred under argon at 55°C for 3 h. The crude 134 product was collected as powder. After recrystalization from methanol, title products 135 (TP-Se) were obtained as a brick-red solid. (Yield, 56.1%). 1H-NMR (300MHz， 136 DMSO-d6), δ (ppm): 8.94 (s, 1H), 8.78 (d, 2H), 8.66 (s, 1H), 8.18 (d, 1H), 8.09 (m, 3H), 137 7.97 (d, 2H), 7.50 (d, 1H), 7.44 (m, 4H), 7.33 (d, 2H), 7.05 (s, 1H), 6.95 (s, 2H), 4.53 (s, 138 2H), 4.39 (s, 3H), 1.29(t, 3H). TOF-MS m/z: calcd. For C35H30N3Se+: 571.6. (1H NMR 139 and TOP mass spectra see Supplementary Fig. 1) 140 2.3. Cell Culture SC RI PT 132 Human hepatocellular liver carcinoma cell line and mouse macrophage cell line 142 (RAW264.7) were maintained following protocols provided by the American-type 143 Tissue Culture Collection. Cells were seeded at a density of 1 × 106 cells mL-1 for 144 confocal imaging in Roswell Park Memorial Institute 1640 medium supplemented with 145 10% fetal bovine serum, NaHCO3 (2 g/L), and 1% antibiotics (penicillin/streptomycin, 146 100 U/ml). Cultures were maintained at 37 °C under a humidified atmosphere 147 containing 5% CO2. 148 3. Results and discussion 149 3.1. Investigation of detecting mechanism TE D M AN U 141 As shown in Scheme 1, structure of TP-Se was confirmed by time-of-flight (TOF) 151 mass analysis. To confirm reaction mechanisms, TOF mass spectra were used to 152 investigate oxidation of TP-Se by ONOO−. A peak corresponding to [TP-Se-I−]+ 153 appeared at m/z = 572.2 (Supplementary Fig. 1e). When 20 equiv. of ONOO− was 154 added to the solution, a peak corresponding to [TP-SeO-I−]+ appeared at m/z=588.1, 155 demonstrating complete oxidation of Se (Scheme 1) (Supplementary Fig. 1f). Mass 156 results confirmed that the Se-containing probe (TP-Se) was converted to corresponding 157 TP-SeO under addition of ONOO−. AC C EP 150 158 Following the ping-pong mechanism , we confirmed a new TP-reversible 159 fluorescent probe (TP-Se) for detection of ONOO− in living cells through rapid 160 photoinduced electron transfer (PET). Fluorescence of TP-Se was quenched as a result 6 ACCEPTED MANUSCRIPT 161 of PET between the modulator and transducer, but Se oxidation prevented PET, 162 “switching on” fluorescence emission (Scheme 1). 163 3.2. Absorption analysis Absorption spectra were obtained using UV-visible spectrophotometer at 25 °C. 165 TP-Se probe (1 ml, 0.01 mM) was added to 1 ml color comparison tubes and 166 investigated through absorption spectroscopy in PBS buffer (50 mM, pH 7.4) with 1% 167 DMSO. Mixtures were equilibrated for 5 min before measurement. All tests were 168 performed in the presence of 0.10 M NaCl to maintain constant ionic strength. As 169 shown in Figure 1a, absorption of TP-Se was 445 nm, showing a blue excitative peak 170 (TP-Se) similar to the red one (TP-Se+ 20 equiv. ONOO−). 171 3.3. Fluorescence analysis M AN U SC RI PT 164 Fluorescence spectra were determined using a spectrofluorometer. TP-Se probe (1 173 ml, 0.01 mM) was added to 1 ml color comparison tubes and investigated through 174 fluorescence spectroscopy in PBS buffer (50 mM, pH 7.4) with 1% DMSO. Mixtures 175 were equilibrated for 5 min before measurement. Fluorescence intensity was measured 176 at λex=565 nm. All tests were performed in the presence of 0.10 M NaCl to maintain 177 constant ionic strength. As shown in Figure 1 (b, c, and d), TP-Se displayed almost no 178 fluorescence in the solution possibly due to weakly radiative deactivation of the excited 179 state through the Se group. Accordingly, emission intensity at 565 nm increased with 180 continuous addition of ONOO− to TP-Se solution and upon excitation at 430 nm and 181 reached its maximum when 25 equiv. of ONOO− was added. UV spectra (Figure 1a) of 182 ONOO− displayed a similar sensing behavior toward addition of more ONOO−. A good 183 linear relationship of emission intensity against ONOO− was observed from 0 equiv. to 184 25 equiv. (inset of Figure 1c), and detection limit (3 σ/k, where σ refers to standard 185 deviation of blank measurement, and k is the slope between fluorescence intensity 186 versus ONOO− concentration.) was as low as 31 nM. Results showed that ONOO− 187 probe possesses good sensitivity for detecting ONOO−. We also monitored 188 fluorescence changes with time when ONOO− (25 equiv.) was added to the solution of 189 TP-Se probe. As shown in Figure 1a, maximum fluorescence intensity was obtained in 190 2 min. All fluorescence tests were performed after 30 min. AC C EP TE D 172 7 ACCEPTED MANUSCRIPT 191 3.4. TP fluorescence measurement TP cross-sections were calculated using TP-induced fluorescence measurement. In 193 the range of 750 nm to 940 nm, maximum TP absorption of cross-sections (δmax) of 194 TP-Se appearing at 865 nm reached 1775 GM (Figure 1e). Emission spectra of TP-Se 195 (10 µ M) with addition of ONOO− (0–25 equiv.) were tested under TP excitation (λex= 196 865 nm) (Figure 1f). Results indicated that TP-Se can function well as TP fluorescent 197 probe for ONOO− in living systems. TP-excited fluorescence titration experiment was 198 also conducted for TP-Se. Under TP excitation (λex= 865 nm), TP-excited 199 fluorescence spectra were enhanced upon addition of ONOO−. SC RI PT 192 Photophysical properties of TP-Se were tested under simulated physiological 201 conditions (Tris buffer, pH 7.4). TP-Se emits at around 565 nm and is excited at 430 202 (OP, Figure 1d) and 865 nm (TP, Figure 1f). 203 3.5. Selectivity toward peroxynitrite at various pH values M AN U 200 We also evaluated the effect of pH on fluorescence. In living cells, different 205 organelles feature varying pH values (Figure 2a). (1) Cytosol works at pH values of 206 6.80 to 7.40, whereas (2) acidic organelles (e.g., lysosomes and endosomes) function in 207 the pH range of 4.5 to 6.0 . In the absence of ONOO−, fluorescence intensity of 208 TP-Se remained unchanged at a pH range of 5.0 to 10.0, indicating that TP-Se was 209 hardly influenced by pH. Meanwhile, upon addition of ONOO−, unchanged and strong 210 fluorescence signals were detected in the pH range of 5.0 to 10.0, which also covers the 211 pH range of physiological environments. Therefore, combined with results of its 212 outstanding response behavior, TP-Se exhibits robust analytical potential for biological 213 applications. 214 3.6. Selectivity of TP-Se and in vitro cytotoxicity studies EP AC C 215 TE D 204 To confirm selectivity of TP-Se for ONOO−, we investigated its responses toward 216 competing ROS (ClO−, O2•−, •OH, 1O2, •NO, H2O2, and ONOO−) and metal ions 217 (including Fe3+, Fe2+, Cu2+, Cu+, Zn2+, and Mg2+). Test solutions of TP-Se (10 µM) in 218 pH 7.4 phosphate-buffered saline (PBS) (containing 1% DMSO) were prepared. 219 Solutions of various test species were prepared by diluting the stock solution with 220 PBS buffer solution. Different anions were prepared with metal ions (10-500 µM) 8 ACCEPTED MANUSCRIPT forming ZnCl2, MgSO4, CuCl2, CuNO3, FeCl3, FeCl2, and ONOO− (10-500 µM). 222 Various ROS (10-100 µM), including ClO−, •OH, O2•−, 1O2, •NO, H2O2, and ONOO−, 223 were prepared according to a previously reported procedure. Results showed that 224 TP-Se exhibited high selectivity for ONOO− and was unperturbed upon addition of 225 other ROS and metal ions (Figures 2b, c). TP-Se displayed an instantaneous response 226 and good photostability (Figure 2b, c, and d). Thus, between TP-Se and intracellular 227 ONOO−, reaction features, including selectivity, reversibility, and instantaneity, endow 228 TP-Se with capability to probe ONOO− dynamically in cells. And in vitro cytotoxicity 229 studies of TP-Se were performed on L929, MCF-7, and Colo 205 cells by MTT assays. 230 These results suggest that TP-Se is a safe and non-toxic probe (Supplementary Fig. 2). 231 3.7. Fluorescence recovery rate with various reducing materials. M AN U SC RI PT 221 Given that various types of antioxidants are contained in cells, an additional test was 233 performed on the probe to determine whether other reducing species will act as 234 interferents. As shown in Figure 3a, TP-Se probe displayed excellent selectivity 235 response to thiols, which are the main antioxidants in vivo at an intracellular 236 concentration of ~5 mM [38, 39]. Average fluorescence recovery rate reached up to 237 ~96.0% for reduction by GSH and L-cysteine. 238 3.8. Reversibility of probe in Solution TE D 232 Fluorescence responses of TP-Se (10 µM) to redox cycles were normalized. TP-Se 240 was oxidized by 1 equiv. of added ONOO−. After 7 min, the solution was treated with 2 241 equiv. of GSH. When fluorescence returned to initial levels, another 1 equiv. of 242 ONOO− was added to the mixture. Redox cycle was repeated four times. ONOO− 243 originates from a peroxynitrite donor, 3-morpholinosydnonimine (SIN-1). All data 244 were acquired from 50 mM PBS (acetonitrile 5% v/v) at pH 7.4 (λex= 445 nm, λem = 565 245 nm). Figure 3b shows that reversible oxidation–reduction cycle can be repeated at least 246 four times with only a modest fluorescence decrement (18% of TP-SeO was bleached 247 during the process). Altogether, results showed that TP-Se is suitable for mitochondrial 248 peroxynitrite-reversible monitoring in living cells. 249 3.9. TP bio-imaging in mitochondria 250 AC C EP 239 To further investigate subcellular localization of TP-Se, HeLa cells and a 9 ACCEPTED MANUSCRIPT commercially available mitochondrial dye (MitoTracker Red FM) were employed for 252 co-localization study. Cells were treated with TP-Se (10 µM) for 30 min and 253 subsequently with ONOO− (20 equiv.) for 15 min and then treated with MitoTracker 254 Red FM (0.6 µM) for 30 min. Repeatability of correlation plot, which describes the 255 distribution between green (Figure 4A-a) and red channels (Figure 4A-b), revealed high 256 co-localization in mitochondria. Results indicated that TP-Se was specifically 257 site-internalized in mitochondria, and it can be used to detect mitochondrial ONOO− 258 under TP excitation in living cells. 259 3.10. Confocal fluorescence images of reversible redox cycles in living cells. SC RI PT 251 We then tested whether the probe can be used to monitor ONOO−-reversible redox 261 cycles in living cells. Living RAW264.7 cells loaded with 10.0 µM TP-Se for 5 min 262 showed only faint fluorescence (Figure 4B-a). However, incubating the same 263 TP-Se-loaded cells with 10.0 µM 3-morpholinosydnonimine (SIN-1), a peroxynitrite 264 donor , resulted in substantial increase in intracellular fluorescence after 10 min as 265 the probe detected oxidative stress (Figure 4B-b). Cells were then treated with the ROS 266 scavenger GSH S-transferase (GST, 250 units/mL). After 10 min, intracellular 267 fluorescence decreased to faint levels as GST reduced cell environment (Figure 4B-c). 268 Addition of a second aliquot of SIN-1 oxidant resulted in another burst of oxidative 269 stress and an increase in intracellular fluorescence (Figure 4B-d). These experiments 270 demonstrated that low levels of intracellular fluorescence, as shown in Figure 4B, were 271 not due to photobleaching or dye leakage. Findings suggest that TP-Se can sense redox 272 cycles through reversible fluorescence responses in living cells. 273 4. Discussion TE D EP AC C 274 M AN U 260 To conclude, we have developed a new Two-photon reversible fluorescent probe 275 (TP-Se), that exhibits good sensitivity and selectivity in monitoring ONOO− oxidation 276 and GSH reduction events under physiological conditions in living cells without 277 interference from other biologically relevant species. The probe effectively hinders 278 influence of autofluorescence in biological systems and poses minimal toxicity to cells. 279 TP-Se can also be used for real-time imaging of living cells. Our results show that 10 ACCEPTED MANUSCRIPT 280 TP-Se probe can be used to visualize mitochondrial peroxynitrite levels with negligible 281 background fluorescence and cellular toxicity. 282 283 Funding This work was supported by Shandong Provincial Natural Science Foundation 285 [ZR2016BL16, ZR2016CL01], China; A project of Shandong Province Higher 286 Educational Science and Technology Program [J17KA120]; Doctor Foundation of 287 Binzhou University [2016Y17, 2016Y02]. 288 Supporting information 290 SC 1 H NMR and Mass spectra see Supplementary Fig. 1. Measurement of TP M AN U 289 RI PT 284 cross-sections, Confocal Imaging, and MTT assay see Supplementary Information. 291 References 293  Y. Xiao, L. Chen, W. Lv, F. Nan, Mitochondrial targeting fluorescent probes for reactive 294 oxygen species, Chemistry of life. 36 (2016) 538-547. 295  Y. Li, X. Xie, X. Yang, M. Li, et al., Two-photon fluorescent probe for revealing drug induced 296 hepatotoxicity via mapping fluctuation of peroxynitrite. Chem. Sci. 8 (2017) 4006–4011. 297  P. Pacher, J.S. Beckman, L. Liaudet, Nitric oxide and peroxynitrite in health and disease, 298 Physiol. Rev. 87 (2007) 315-424. 299  N.B. Surmeli, N.K. Litterman, A.F. Miller, J.T. Groves, Peroxynitrite mediates active site 300 tyrosine nitration in manganese superoxide dismutase. evidence of a role for the carbonate radical 301 anion, J. Am. Chem. Soc. 132 (2010) 17174–17185. 302  L. Liaudet, G. Vassalli, P. Pacher, Role of peroxynitrite in the redox regulation of cell signal 303 transduction pathways, Front Biosci. 14 (2009) 4809–4814. 304  H. Kawasaki, K. Ikeda, A. Shigenaga, T. Baba, et al., Mass spectrometric identification of 305 tryptophan nitration sites on proteins in peroxynitrite-treated lysates from PC12 cells, Free Radic. 306 Biol. Med. 50 (2011) 419–427. 307  T. Sawa, M.H. Zaki, T. Okamoto, T. Akuta, et al.. Protein S-guanylation by the biological AC C EP TE D 292 11 ACCEPTED MANUSCRIPT signal 8-nitroguanosine 3',5'-cyclic monophosphate, Nat. Chem. Biol. 3 (2007) 727–735. 309  R. Wang, L. Chen, P. Liu , Q. Zhang, Y. Wang, Sensitive near-infrared fluorescent probes for 310 thiols based on Se-N bond cleavage: imaging in living cells and tissues, Chemistry. 18 (2012) 311 11343-11349. 312  F. Yu, P. Li, P Song, B. Wang, J. Zhao, Facilitative functionalization of cyanine dye by an 313 on-off-on fluorescent switch for imaging of H2O2 oxidative stress and thiols reducing repair in 314 cells and tissues, Chemical Communications. 48 (2012) 4980-4982. 315  L.Y. Niu, 316 fluorescent sensor for highly selective detection of glutathione over cysteine and homocysteine, 317 Journal of the American Chemical Society. 134 (2012) 18928-18931. 318  H.L. Min, J.H. Han, J.H. Lee, H.G. Choi, C. Kang, Mitochondrial thioredoxin-responding 319 off-on fluorescent probe, J. Am. Chem. Soc. 134 (2012) 17317–17319. 320  G. Ferrersueta, R. Radi. Chemical biology of peroxynitrite: kinetics, diffusion, and radicals. 321 ACS Chemical Biology. 4 (2009)161-177. 322  P. Pacher, J. S. Beckman, L.Liaudet, Nitric oxide and peroxynitrite in health and disease. 323 Physiol. Rev. 87 (2007) 315–424. 324  R. Radi, A. Cassina, R. Hodara, C. Quijano, L. Castro, Peroxynitrite reactions and formation 325 in mitochondria, Free Radical Biology &Medicine. 33 (2002) 1451-64. 326  E. Novo, M. Parola, Redox mechanisms in hepatic chronic wound healing and fibrogenesis, 327 Fibrogenesis & Tissue Repair. 1 (2008) 5. 328  B. Morgan, D. Ezeriņa, T.N.E. Amoako, J. Riemer, M. Seedorf, Multiple glutathione disulfide 329 removal pathways mediate cytosolic redox homeostasis, Nature Chemical Biology. 9 (2013) 330 119-125. 331  S.L. Chang, G. Masanta, H.J. Kim, H.H. Ji, H.M. Kim. Ratiometric detection of 332 mitochondrial thiols with a two-photon fluorescent probe, Journal of the American Chemical 333 Society. 133 (2011) 11132-11135. 334  C.C. Winterbourn. Reconciling the chemistry and biology of reactive oxygen species, Nature 335 Chemical Biology. 4 (2008) 278-286. 336  J.T. Rotruck, A.L. Pope, H.E. Ganther, A.B. Swanson, et al. Selenium: Biochemical role as a 337 component of glutathione peroxidase, Science. 179 (1973) 588–590. RI PT 308 AC C EP TE D M AN U SC Y.S. Guan, Y.Z. Chen, L.Z. Wu, C.H. Tung, BODIPY-based ratiometric 12 ACCEPTED MANUSCRIPT 338  H. Masumoto, R. Kissner, W.H. Koppenol, H. Sies. Kineticstudy of the reaction of ebselen 339 with peroxynitrite, Febs Lett. 398 (1996) 179–182. 340  X. Chen, T. Pradhan, F. Wang, J.S. Kim, J. Yoon. Fluorescent chemosensors based on 341 spiroring-opening of xanthenes and related derivatives, Chemical Reviews. 112 (2012) 1910-56. 342  D.J. Stephens, V.J. Allan, Light microscopy techniques for live cell imaging, Science. 343 (2003) 82-86. 344  M.P. Murphy, A. Holmgren, N. Larsson, et al., Unraveling the biological roles of reactive 345 oxygen species, Cell Metabolism. 13 (2011) 361−366. 346  J.J. Gao, K.H. Xu, B. Tang, L.L. Yin, G.W. Yang, Selective detection of superoxide anion 347 radicals generated from macrophages by using a novel fluorescent probe, Febs Journal. 274 (2010) 348 1725-1733. 349  W. Zhang, P. Li, F. Yang, X. Hu, et al., Dynamic and reversible fluorescence imaging of 350 superoxide anion fluctuations in live cells and in vivo, J. Am. Chem. Soc. 135 (2013) 351 14956−14959. 352  J.H. Lee, C.S. Lim, Y.S. Tian, J.H. Han, B.R. Cho, A two-photon fluorescent probe for thiols 353 in live cells and tissues, Journal of the American Chemical Society. 132 (2010) 1216-1217. 354  C. Chung, D. Srikun, C.S. Lim, C.J. Chang, B.R. Cho. A two-photon fluorescent probe for 355 ratiometric imaging of hydrogen peroxide in live tissue, Chemical Communications. 47 (2011) 356 9618-9620. 357  S.L. Chang, G. Masanta, H.J. Kim, H.H. Ji, H.M. Kim, 358 mitochondrial thiols with a two-photon fluorescent probe, Journal of the American Chemical 359 Society. 133 (2011) 11132-11135. 360  C.C. Winterbourn, Reconciling the chemistry and biology of reactive oxygen species, Nat. 361 Chem. Biol. 4 (2008) 278. 362  J.T. Rotruck, A.L. Pope, H.E. Ganther, A.B. Swanson, Selenium: biochemical role as a 363 component of glutathione peroxidase, Science. 179 (1973) 588-590. 364  H. Masumoto, R. Kissner, W.H. Koppenol, H. Sies, Kinetic study of the reaction of ebselen 365 with peroxynitrite, Febs Letters. 398 (1996) 179-182. 366  F. Miao, W.J. Zhang, Y.M. Sun, R.Y. Zhang, Y. Liu, F.Q. Guo, G.F. Song, M.G. Tian, X.Q. Yu, 367 Novel fluorescent probes for highly selective two-photon imaging of mitochondria in living cells, Ratiometric detection of AC C EP TE D M AN U SC RI PT 300 13 ACCEPTED MANUSCRIPT Biosens. Bioelectron. 55 (2014) 423–429. 369  D.X. Li, X. Sun, J.M. Huang, Q. Wang, Y. Feng, M. Chen, X.M. Meng, M.Z. Zhu, X. Wang, 370 A carbazole-based “turn-on” two-photon fluorescent probe forbiological Cu2+ detection vis 371 Cu2+-promoted hydrolysis, Dyes Pigments. 125 (2016) 185–191. 372  D.X. Li, Y. Feng, J.Z. Lin, M. Chen, S.X. Wang, X. Wang, H.T. Sheng, Z.L. Shao, M.Z. Zhu, 373 X.M. Meng, A mitochondria-targeted two-photon fluorescent probe for highly selective and rapid 374 detection of hypochlorite and its bio-imaging in living cells, Sens. Actuators B. 222 (2016) 375 483–491. 376  Y. Feng, D. Li, Q. Wang, S. Wang, X. Meng, Z. Shao, et al., A carbazole-based 377 mitochondria-targeted two-photon fluorescentprobe for gold ions and its application in living cell 378 imagingYan, Sensors and Actuators B. 225 (2016) 572–578. 379  J.T. Hou, M.Y. Wu, K. Li, J. Yang, K.K. Yu, Y.M. Xie, X.Q. Yu, Mitochondria-targeted 380 colorimetric and fluorescent probes for hypochlorite and their applications for in vivo imaging, 381 Chem. Commun. 50 (2014) 8640–8643. 382  H.D. Xiao, K. Xin, H.F. Dou, G. Yin, Y.W. Quan, R.Y. Wang, A fast-responsive 383 mitochondria-targeted fluorescent probe detecting endogenous hypochlorite in living RAW 264.7 384 cells and nude mouse, Chem. Commun. 51 (2015)1442–1445. 385  A. Pastore, F. Piemonte, M. Locatelli, A. Lo Russo, et al., Determination of blood total, 386 reduced, and oxidized glutathione in pediatric subjects. Clinical Chemistry. 47 (2001) 1467-1469. 387  F. Yu, P. Li, G. Li, G. Zhao, T. Chu, et al., A Near-IR reversible fluorescent probe modulated 388 by selenium for monitoring peroxynitrite and imaging in living cells, J. Am. Chem. Soc. 133 (2011) 389 11030–11033. 390  N. Ashki, K.C. Hayes, F. Bao, The peroxynitrite donor 3-morpholinosydnonimine induces 391 reversible changes in electrophysiological properties of neurons of the guinea-pig spinal 392 cord, Neuroscience. 156 (2008) 107-117. AC C EP TE D M AN U SC RI PT 368 393 394 395 396 397 14 ACCEPTED MANUSCRIPT Figure 1. (a) Absorption spectra of TP-Se (1 ml, 0.01 mM) in the absence and presence of ONOO− 399 (20 equiv.) in PBS buffer (50 mM, pH 7.4) with 1% DMSO. (b) Time-dependent fluorescence 400 intensity changes of TP-Se (1 ml, 0.01 mM) with addition of ONOO− (25 equiv.) in PBS buffer. (c) 401 The relationship between fluorescence intensity and ONOO− concentrations and under UV light 402 (λex= 430 nm). (d) Fluorescence spectra of TP-Se (1 ml, 0.01 mM) in the presence of increasing 403 amount of ONOO− (0–25 equiv.) in PBS buffer (50 mM, pH 7.4) with 1% DMSO, λex= 430 nm). (e) 404 TP absorption of TP-Se cross-sections (10 µM) with addition of ONOO− (25 equiv.). (f) Emission 405 spectra of TP-Se (10 µM) with addition of ONOO− (0-25 equiv.) under TP excitation (λex= 865 nm). RI PT 398 SC 406 Figure 2. (a) Fluorescence intensity change in TP-Se (1 µM) with addition of ONOO− (15 µM) 408 under different pH values (5.0, 6.0, 7.0, 7.4, 8.0, 9.0, and 10.0) by using universal buffer solution 409 (0.1 mM citric acid, 0.1 M KH2PO4, 0.1 M Na2B4O7, 0.1 M Tris, 0.1 M KCl) with 1% DMSO. 410 λex/λem = 445/565 nm. (b and c) Fluorescence responses of TP-Se (10 µM) to various metal ions. 411 Data were acquired in 0.03 M Tris, pH 7.4, with λex = 430 nm. (d) Fluorescence responses of 412 TP-Se (10 µM) to various ROS and nitrogen species. Data were acquired in 0.03 M Tris, pH 7.4, 413 with λex = 430 nm. TE D 414 M AN U 407 Figure 3. (a) Fluorescence recovery rate with various reducing materials. TP-Se (10.0 µM) was 416 oxidized by addition of 1 equiv. of ONOO−. Then, the solution was treated with various reducing 417 materials for 20 min: 1. metallothionein (80.0 µM); 2. vitamin C (80 µM); 3. L-cysteine (25.0 µM); 418 4. GSH (25.0 µM); 5. vitamin E (80 µM); 6. uric acid (80 µM); 7. tyrosine (80 µM); 8. histidine (80 419 µM). Fluorescence recovery percentage is defined as (F–F0)/F ×100%, here F corresponds to 420 fluorescence intensity of TP-SeO (10.0 µM), and F0 is fluorescence intensity of the probe after 421 addition of reducing material. (b) Normalized fluorescence responses of TP-Se (10 µM) to redox 422 cycles. TP-Se was oxidized by 1 equiv. of added ONOO− (blue lines). After 7 min, the solution was 423 treated with 2 equiv. of GSH (red lines). Red lines represent normalized intensities obtained after 424 treatment with 2 equiv. of GSH. AC C EP 415 425 426 Figure 4. Confocal fluorescence images of HeLa cells. Confocal fluorescence images of HeLa 427 cells after treatment with TP-Se (10 µM) for 30 min and subsequently with ONOO− (20 equiv.) for 15 ACCEPTED MANUSCRIPT 15 min and then with MitoTracker Red FM (0.6 µM) for 30 min. (A-a) Green emission (530–570 429 nm), λex= 865 nm; (A-b) red emission (580–665 nm), λex= 644 nm. Confocal fluorescence images 430 of reversible redox cycles in living RAW264.7 cells. (B-a) RAW264.7 cells loaded with 10.0 µM 431 TP-Se for 5 min. (B-b) Dye-loaded cells treated with 10.0 µM SIN-1 for 10 min. (B-c) Dye-loaded, 432 SIN-1-treated cells incubated with GST for 10 min. (B-d) Cells exposed to a second dose of SIN-1 433 for an additional 10 min. (B-e) Dye-loaded, SIN-1-treated cells incubated with a second dose of 434 GST (250 units/mL) for 10 min. (B-f) Bright-field image of (B-a). (B-g) Relative the fluorescent 435 intensity of TP-Se-labeled cells in panels B(a−e). Cells shown are representative images from 436 replicate experiments (n = 5). AC C EP TE D M AN U SC RI PT 428 16 ACCEPTED MANUSCRIPT 0.3 TP-Se TP-Se+20 equiv.ONOO¯ 400 b 350 300 0.2 0.1 0 380 400 420 440 460 480 250 200 150 100 0 500 0 Wavelength(nm) FL Intensity(a.u.) FL Intensity(a.u.) 25 24.5 0 1 2 3 4 [ONOO ] μM 2100 900 600 300 0 760 780 800 825 840 865 880 895 920 940 AC C EP Wavelength(nm) 350 300 250 200 150 100 50 0 520 6 TE D δ/GM 1500 1200 5 TP-Se+ONOO¯ 1800 400 M AN U 24 23 e d 2 R = 0.9872 23.5 120 180 240 300 360 420 480 SC y = 0.3964x + 23.527 25.5 60 Time(s) 545 570 595 620 645 Wavlength(nm) f TP Fluorescence Intensity 26 c ← ONOO¯ added 50 RI PT 0.4 FL Intensity(a.u.) a Absorbance(a.u.) Figure 1. 400 350 300 250 200 150 100 50 0 525 545 565 Wavelength(nm) 585 ACCEPTED MANUSCRIPT Figure 2. 400 b 350 300 TP-Se+ONOO¯ 250 TP-Se 200 150 100 50 FL Intensity(a.u.) 400 350 20μM 50μM 100μM 250 500μM 200 150 100 6 c 400 FL Intensity(a.u.) 5 300 20μM 250 50μM 100μM 7 pH 8 9 0 10 Cu²⁺ d FL Intensity(a.u.) 10μM 200 150 100 50 0 Cu⁺ 350 0 min 20 min 40 min 60 min 300 250 200 150 100 50 0 O₂˙ˉ ·OH NO ¹O₂ ONOOˉ H₂O₂ OClˉ Fe²⁺ O₂˙ˉ ·OH AC C EP TE D M AN U OClˉ Fe³⁺ RI PT 50 0 350 10μM 300 Zu²⁺ ONOOˉ SC FL Intensity(a.u.) a NO ¹O₂ Mg²⁺ ONOOˉ H₂O₂ ACCEPTED MANUSCRIPT 120 b 100 Normalized Intensity 80 60 40 20 0 1 2 3 4 5 6 7 8 400 350 300 250 200 150 100 50 0 1 7 14 21 28 35 42 49 56 Cycle times(min) AC C EP TE D M AN U SC Fluorescence Recovery Percentage(%) a RI PT Figure 3. ACCEPTED MANUSCRIPT Figure 4. M AN U SC RI PT A AC C EP TE D B Fluorescence(a.u.) g 90 80 70 60 50 40 30 20 10 0 a b c d e f ACCEPTED MANUSCRIPT Highlights 1. We report a two-photon fluorescent probe (TP-Se) for optical imaging. 2. TP-Se can monitor reversibly changes in ONOO−/GSH levels in mitochondria. RI PT 3. TP-Se will lead to significant development on redox events in cellular redox AC C EP TE D M AN U SC regulation.