Visualizing Endogenous Sulfur Dioxide Derivatives in Febrile-Seizure

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Visualizing Endogenous Sulfur Dioxide Derivatives in Febrile SeizureInduced Hippocampal Damage by a Two-Photon Energy Transfer Cassette Sheng Yang, Xidan Wen, Xiaoguang Yang, Yuan Li, Chongchong Guo, Yibo Zhou, Heping Li, and Ronghua Yang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04355 • Publication Date (Web): 26 Nov 2018 Downloaded from http://pubs.acs.org on November 26, 2018

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Analytical Chemistry

Visualizing Endogenous Sulfur Dioxide Derivatives in Febrile Seizure-Induced Hippocampal Damage by a Two-Photon Energy Transfer Cassette Sheng Yang,† Xidan Wen,† Xiaoguang Yang,† Yuan Li,‡ Chongchong Guo,† Yibo Zhou,† Heping Li,† and Ronghua Yang†,* †

School of Chemistry and Biological Engineering, Hunan Provincial Key Laboratory

of Materials Protection for Electric Power and Transportation, Changsha University of Science and Technology, Changsha, 410114, P. R. China ‡

State Key Laboratory of Chemo/Biosensing and Chemometrics, College of

Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, P. R. China

*To whom correspondence should be addressed:

E-mail: [email protected] Fax: +86-731-8882 2523

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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

ABSTRACT: Febrile seizure (FS), a frequently encountered seizure disorder in pediatric populations, can cause hippocampus damage. It has been elucidated that sulfur dioxide (SO2) content is overproduced during the development of FS and related brain injury. Thus, in situ monitoring the level of endogenous SO2 in FS-related models is helpful to estimate the pathogenesis of FS-induced brain injury, but effect detection method remains to be explored. Herein, we developed a two-photon energy transfer cassette based on acedan-anthocyanidin scaffold, TP-Ratio-SO2, enabling to achieve this purpose. TP-Ratio-SO2 specifically responds to SO2 derivatives (HSO3-/SO32-) in ultrafast fashion (less than 3s), and HSO3-/SO32can be sensitively determined with a detection limit of 26 nM. Moreover, it exhibits significant changes in two well-resolved fluorescence emission (∆λ = 140 nm) by reacting with HSO3-/SO32-, behaving as a ratiometric fluorescent sensor. Importantly, ratiometric imaging of endogenous SO2 derivatives generation in hyperpyretic U251 cells and as well as in rat model of FS-treated hippocampus damage were successfully carried out by TP-Ratio-SO2, demonstrating that it may be a promising tool for studying the role of SO2 in FS-associated neurological diseases.

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INTRODUCTION Febrile seizure (FS) is a frequently encountered seizure disorder in pediatric populations, occurring in 2–4% of children between the ages of 6 months and 5 years.1 Epidemiological studies have suggested that recurrent FS may be implicated in some long-term consequences, such as a decreased seizure threshold, memory problems, and learning difficulties.2 Moreover, FS can cause neuronal damage and enhanced cytogenesis in the hippocampus,3 which is one of the most common neuropathological findings of temporal lobe epilepsy.4 It has been reported that sulfur dioxide (SO2) as an important gasotransmitter,5 equilibrating with aqueous sulfites/bisulfites (HSO3-/SO32-) in biosystems, is involved in the development of FS and associated brain injury.6 Obviously, monitoring the level of endogenous SO2 derivatives with efficient methods is crucial to elucidate the pathogenesis of FS-induced brain injury. Some conventional analytical techniques such as electrochemical measurement,7 chromatography,8 spectrophotometry,9 flow injection analysis,10 piezoelectric sensor,11 and capillary electrophoresis,12 are available for the determination of SO2 derivatives in vitro. Nevertheless, their further applications in organisms were restricted by sample destruction, low operability, and time-consuming pretreatment. On the other hand, fluorescent probe-based imaging has emerged as an useful methodology for real time and in situ tracking of biologically relevant species in vivo.13 Accordingly, a number of fluorescent probes for SO2 derivatives have been developed hitherto, mostly through reaction approach utilizing HSO3-/SO32--mediated levulinate 3



cleavage14,15 and nucleophilic addition to aldehyde16-18 or hemicyanine moiety.19-24 Several challenges in designing fluorescent probes for SO2 derivatives detection in living biological systems are how to guarantee (i) enough sensitivity to potentially detect endogenous SO2 derivatives,25-27 (ii) instantaneous response to tackle quick SO2 metabolism,28,29 (iii) high selectivity to overcome the interference from biothiols, proteases or esterases in complicated biosystem,30 (iv) excellent two-photon property with fixed excitation maximum and ratiometrical signal changes with large emission shift beneficial to deep-tissue and high resolution imaging.31-34 Unfortunately, the aforementioned probes hardly fulfill all of these criteria, impeding their further applications to tackle biological issues. Until now, no fluorogenic probe was available to investigate the role of SO2 in FS-induced brain injury. To address this issue and as our continuous research on fluorogenic probes for signaling molecules including hydrogen sulfide (H2S),35 hydrogen peroxide (H2O2),36 nitroxyl (HNO),37 and hydrogen polysulfide (H2Sn).38 Herein, we designed and synthesized an efficient ratiometric two-photon fluorescence probe for SO2 derivatives, TP-Ratio-SO2, enabling to efficiently assay and image SO2 derivatives in FS-induced brain injury. As depicted in Scheme 1, TP-Ratio-SO2 was composed of an acedan–anthocyanidin scaffold as two-photon energy transfer dyad. Upon two-photon excitation, it displays characteristic emission of anthocyanidin derivative at 640 nm due to the two-photon excited fluorescence resonance energy transfer (TP-FRET) process.39 In the presence of SO2 derivatives, nucleophilic addition of HSO3-/SO32interrupts the conjugation structure of anthocyanidin derivative to impede the 4


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TP-FRET process, resulting in disappearance of anthocyanidin emission and recovery of acedan fluorescence emission at 500 nm. TP-Ratio-SO2 exhibited specific and ultrafast response to SO2 derivatives in less than 3s, and HSO3-/SO32- could be sensitively determined with a detection limit of 26 nM. Moreover, its reaction with SO2 derivatives causes significant variations in fluorescence intensity ratio (I640/I500) with a large emission shift (∆λ = 140 nm). Importantly, the proposed probe has demonstrated its capability for visualizing endogenous SO2 derivatives in hyperpyretic U251 cells and rat model of FS-treated hippocampus damage by two-photon ratiometric fluorescence imaging. To the best of our knowledge, this is the first attempt to develop molecular tool for exploring the role of SO2 in FS-associated neurological diseases.

EXPERIMENTAL SECTION Synthesis of TP-Ratio-SO2. Firstly, the carboxylated anthocyanidin derivative 3 and piperazine-modified acedan derivative 5 were successively synthesized according to the synthetic procedure as depicted in Scheme 2. Their detailed preparations are attached to the Supporting Information. Subsequently, Compound 3 (100.3 mg, 0.285 mmol), 5 (107.6 mg, 0.285 mmol), EDC (60 mg, 0.3 mmol) and HOBt (48 mg, 0.3 mmol) were dissolved in CH2Cl2 (4.0 mL). The mixture was stirred under N2 atmosphere for overnight at the room temperature. The resulting mixture was extracted with saturated NaHCO3, dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The resulting residue was purified by column chromatography (CH2Cl2/MeOH, 20:1) on silica gel to give the final product 5



TP-Ratio-SO2 as a dark red solid (106.7 mg, yield: 52.6%).1H NMR (400 MHz, DMSO): δ 1.26-1.34 (m, 6H), 1.73(s, 2H), 1.87 (s, 2H), 2.02 (d, J = 8.0 Hz, 2H), 2.61 (s, 2H), 2.96 (s, 4H), 3.61 (d, J = 4.0 Hz, 9H), 3.70 (s, 3H), 4.23-4.33(m, 2H), 6.70 (s, 1H), 6.96 (d, J = 15.6 Hz, 2H), 7.14 (s, 1H), 7.22-7.26 (m, 1H), 7.64 (d, J = 8.0 Hz 1H), 7.77-7.81(m, 3H), 7.87 (d, J = 4.0 Hz, 1H), 8.06 (d, J = 8.0 Hz, 1H), 8.37 (s, 1H), 8.40 (d, J = 11.8 Hz, 1H).

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C NMR (100 MHz, DMSO): δ 197.34, 174.93, 170.87,

167.14,165.22, 157.19, 155.78, 153.98, 147.27,145.37, 137.69, 131.25, 131.05, 130.23, 126.10, 124.87, 124.45, 120.66, 117.07, 115.71, 115.45, 114.00, 104.98, 96.33, 58.51, 52.49, 48.76, 45.33, 31.74, 31.62, 30.31, 29.47, 29.14, 26.80, 25.66, 25.54, 22.54, 14.40,12.86. EI-MS (m/z): calcd for C44H48N5O4+: 710.88; found: 710.4. Spectrophotometric Experiments. Stock solution (5 mM) of the probe was prepared by dissolving TP-Ratio-SO2 in DMSO. Both the UV-vis absorption and fluorescence measurements were conducted in phosphate buffer solution with DMSO as the co-solvent (H2O/DMSO = 7:3, v/v). For fluorogenic assay in aqueous solution, 500 μL of buffer containing 1 μM TP-Ratio-SO2 was first introduced to a quartz cell and placed for 5 min. Following the additions of 10 μL different concentrations of Na2SO3 or other competition species, the mixture solutions were kept at room temperature for 5 min and then the fluorescence spectra were then recorded (for one-photon: λex = 390 nm, λem = 450-720 nm, for two-photon: λex = 760 nm, λem = 450-700 nm). Imaging of SO2 Derivatives in Living Cells. For imaging of exogenous SO2, the U251 cells were pretreated with TP-Ratio-SO2 (1.0 μM) for 30 min before washed three times with PBS, then incubated with different concentrations of Na2SO3 solution 6


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for 5 min. In order to monitor the generation of endogenous SO2 in U251 cells under hyperpyretic treatment, the cells were incubated at the temperature of 45 ℃ for 30 min. Another group was pretreated with HDX (an endogenous SO2 inhibitor, 100 nmol) before loading with probe at the temperature of 45 ℃ for 30 min. As a negative control, the third group’s U251 cells were incubated at the temperature of 37 ℃ for 30 min. After rinsing with PBS buffer, these cells were exposed to imaging experiment on the two-photon confocal microscope. Imaging of Mouse Liver Slices and Samples of Febrile Seizure Rat Model. Mouse liver tissue slices were prepared from the 2-weeks-old rat (SD) according to the protocol No. SYXK (Xiang) 2013-0001, approved by Laboratory Animal Center of Hunan. The slices were stained with 5 μM P-Ratio-SO2 for 30 min, then incubated with different concentrations of Na2SO3 solution for 5 min. After rinsing with PBS buffer, the slices were subjected to imaging analysis similar as cellular ones. The rat model of FS induced hippocampus injury was established following a reported approach. Rats were randomly divided into third groups: control group, FS group, and FS with HDX group. Briefly, the control rat was put into 37 ℃ thermostat water bath for 5 h. The rats in the other two groups were put into 45.2 ℃ water until a seizure occurred. Water-immersion was carried out 10 times, once every 2 days. For rat in FS with HDX group, 3 mg/kg HDX was administered intraperitoneally. An equal volume of normal saline was intraperitoneally injected into rats in the control and FS groups. After last time water immersion, all the rats were euthanized, and their hippocampus tissue samples were harvested. The tissue slices were cut into 400 μM in 7



size using a vibrating-blade microtome, and then loaded with the 5 μM TP-Ratio-SO2 for 1 h. The residual probe was removed by washing three times using PBS buffer before the imaging by two photon confocal microscope. Two-photon imaging pictures were obtained from the green channel (490-520 nm) and red channel (610-660 nm) with two-photon excitation wavelength of 760 nm. The ratiometric imaging patterns were analyzed by Image J software.

RESULTS AND DISCUSSION Design and Synthesis of TP-Ratio-SO2. We previously reported the first-generation TP-FRET-based ratiometric two-photon fluorescence probe for SO2 derivatives,40 composed of a well-known two-photon fluorophore acedan moiety and a HSO3-/SO32--responsive merocyanine-analogue dye as donor and acceptor of energy transfer dyad, respectively. Unfortunately, deficient TP-FRET efficiency and relatively sluggish reaction kinetics constrains its potential for sensitively and rapidly measuring endogenous release of SO2 derivatives during FS-induced neuronal damage in the hippocampus. To overcome these weaknesses, we intended to rationally design TP-Ratio-SO2 as the improved one by substituting merocyanine moiety with anthocyanidin. Anthocyanidin derivative was chosen as the new energy acceptor in accordance with the following reasons: Firstly, it shows appropriate absorption spectrum matching with two-photon emission spectra of TP (Figure S1), indicating that the acedan-anthocyanidin scaffold would fully fluoresce at the characteristic emission of acceptor moiety due to effective two-photon excitation energy transfer process. Secondly, it is well established that anthocyanidin derivatives are robust 8


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HSO3-/SO32- receptors,28,31 as demonstrated by the dramatic decrease in absorbance of acceptor moiety 3 after treatment with Na2SO3 (curve b of Figure S1). To support the design rationality, density functional theory (DFT) calculations were carried out (Figure S2). For TP-Ratio-SO2, the HOMO-LUMO energy gap of the anthocyanidin moiety (ΔE = 2.64 eV) is lower than that of the acedan moiety (ΔE = 4.02 eV), facilitating the intramolecular energy transfer from acedan to anthocyanidin. Nonetheless, after treatment with Na2SO3, both HOMO and LUMO orbitals show that the π electrons are not located around the whole acceptor moiety, indicating that the conjunction structure of anthocyanidin is destroyed, moreover, its energy gap (ΔE = 5.62 eV) is bigger than that of the energy donor (ΔE =3.94 eV), thus the TP-FRET process would be impeded.37 With the design concept in mind, we efficiently synthesized TP-Ratio-SO2 according to the synthetic procedure as depicted in Scheme 2. The carboxylated anthocyanidin derivative 3 was firstly synthesized through three-step reaction from 6-amino-1,2,3,4-tetrahydro-1-naphthalenone. Piperazine-modified acedan derivative 5 was then synthesized through N-Boc deprotection of the condensation product of acedan TP with 4-(N-Boc-amino) piperidine. Subsequently, amidation reaction of compound 3 and 5 was performed in the presence of EDC and HOBt to give the final probe TP-Ratio-SO2. Its structure was confirmed by 1H NMR,

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C NMR, and MS

analysis, and the corresponding results were attached to the Supporting Information. Spectroscopic Properties and Response Performances of TP-Ratio-SO2. With the probe in hand, we start to test its photophysical property and response capacity to 9



HSO3-/SO32-. As shown in Figure 1A, TP-Ratio-SO2 exhibited absorption peaks at 378 nm and 580 nm, characteristic of acedan and anthocyanidin derivative respectively. Upon addition of Na2SO3, the absorption peak gradually attenuated concomitant with a colour change from bluish violet to colourless (inset of Figure 1A), demonstrating that TP-Ratio-SO2 could response to SO2 derivates. Moreover, the appearance of new MS peak at m/z 791.4 provide the evidence of reaction between TP-Ratio-SO2 and Na2SO3 (Figure S3). As expected, TP-Ratio-SO2 exhibits strong fluorescence emission at 640 nm but a minor band at around 500 nm under two-photon excitation (ex = 760 nm). Moreover, the emission intensity of TP-Ratio-SO2 is 3.1-fold higher than that of the free acceptor 3 under the same irradiation condition, revealing that TP-FRET process from the acedan donor to the anthocyanidin-analogue acceptor availably occurs. However, the maximum emission wavelength significantly shifts from 640 nm to 500 nm after the addition of Na2SO3, concomitant with observable fluorescence tint change from red to green (inset of Figure 1B). Obviously, such large emission shift (∆λ = 140 nm) would be beneficial for high-resolution fluorescence imaging. Noteworthy, both TP-Ratio-SO2 and its reaction product with Na2SO3 exhibit excellent two-photon excitation action cross-section (Φδ) about 100 GM with fixed two-photon excitation maximum at 760 nm (Figure S4), which is more suitable for two-photon imaging than the reported ICT-based ratiometric ones.31-34 To evaluate the sensing performances of TP-Ratio-SO2, concentration-dependent fluorometric titration of TP-Ratio-SO2 toward Na2SO3 under the one-photon 10


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excitation (390 nm) were then carried out. As shown in Figure 2A, increasing concentrations of Na2SO3 elicited significant decrease in emission peak at 640 nm and simultaneously a hypsochromic center around 500 nm gradually raised with a clear iso-emission point at 600 nm. With the increasing concentration of Na2SO3 (0-400 μM), the fluorescence intensity ratio (F500/F640) varied from 0.27 to 7.35 with a 27-fold enhancement in the signal to background ratio (S/B = (F500/F640)n/(F500/F640)0), as depicted in Figure 2B. Moreover, the S/B value is proportional to the concentrations of Na2SO3 in the range 0 to 50 μM (inset in Figure 2B) and the detection limit for Na2SO3 was calculated to be 26 nM (3σ/slope). Comparative to the first-generation one, broader response range and improved sensitivity would make TP-Ratio-SO2 more potential for quantitative detection of SO2 derivatives. The response kinetics of TP-Ratio-SO2 toward Na2SO3 were then tested. As depicted in Figure 3, the emission intensity ratio, F500/F640, immediately elevated and reached a maximum equilibrium within 3 second after the addition of 30-200 μM Na2SO3 solution. Such an ultrafast fashion is beneficial to real-time detection. In the absence of Na2SO3, no detectable change can be observed with time, manifesting that the free probe maintains stability under aqueous solution. The pH effect on TP-Ratio-SO2 and its in response to SO2 derivates were subsequently investigated (Figure S5). The emission intensity ratio of TP-Ratio-SO2 is stable in the broad pH range (3.0-10.0) and an obvious enhancement of ratiometric signals was found after it treated with Na2SO3 within the normal physiological ranges, suggesting TP-Ratio-SO2 is stable and functions properly at a biologically relevant 11



pH condition. To evaluate the probe specificity, some common anions (F−, Cl−, Br−, I−, AcO−, P2O5−, HCO3−, CN−, SO42−, NO2−), representative reactive oxygen species (H2O2), and other reactive sulfur species (HSO3−, S2O32−, HS−, Cys, Hcy, and GSH) were treated with TP-Ratio-SO2. Figure 4 depicted that no significant fluorescence intensity ratio change could be observed in the presence of above bio-relevant molecules other than SO32-/HSO3-, indicating that TP-Ratio-SO2 possesses selectivity for SO2 derivates over other bio-relevant species. Furthermore, competition experiments revealed that above bio-relevant molecules would not and hardly interfere with specific response of TP-Ratio-SO2 toward SO2 derivates, which is helpful for the probe to specifically capture SO2 derivates in complicated biological environment. Two-Photon Bioimaging of Endogenous SO2 in Hyperpyretic U251 Cells. Inspired by the favorable response performances of probe TP-Ratio-SO2 to SO2 derivates under aqueous conditions, we further tested its capability for ratiometrically visualizing SO2 derivates in human glioma cell line (U251). As shown in Figure S6, U251 cells staining with 1 μM TP-Ratio-SO2 fluoresce in red channel (610−660 nm), together with negligible signal through green channel (490−520 nm). However, the green fluorescence gradually brightens in the contrast cells pretreated with incremental concentrations of Na2SO3, along with decrease of emission in the red channel. Moreover, red-to-green fluorescence signal change was quickly visualized in only 2 min (Figure S7). In addition, TP-Ratio-SO2 was not toxic to the U251 cells during the concentration ranging from 0 to 10 μM via the standard MTT assay (cell 12


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viability:＞95%, Figure S8). These pattern results revealed that TP-Ratio-SO2 was cell-permeable and could be utilized in ratiometric fluorescence visualization of variations in concentrations of intracellular SO2 derivates. It is well established that SO2 can be endogenously produced from aspartate aminotransferase (AAT)-catalyzed metabolism of sulfur-containing small molecules.41 To demonstrate that SO2/ATT system is associated with the development of FS, TP-Ratio-SO2 was further used to investigate the fluctuation of endogenous SO2 derivates in U251 cells under hyperpyretic treatment (Figure 5). In comparison with the normal probe-treated ones, the hyperpyretic U251 cells incubated with TP-Ratio-SO2 showed apparently enhancive fluorescence in green channels alongside with signal attenuation in red channels. By contrast, no obvious change in fluorescence emission ratio can be found when probe-incubated hyperpyretic U251 cells were pretreated with L-aspartate-hydroxamate (HDX), an inhibitor of the AAT enzyme responsible for endogenous SO2 production.42 Moreover, these fluorescence imaging results were further verified by flow cytometry analysis. In addition, control experiment revealed that TP-Ratio-SO2 and its response performance were both temperature-independent (Figure S9), confirming that the fluorescence ratio changes were indeed initiated from endogenously generated SO2 derivates rather than temperature change. These results strongly suggest that increased endogenous SO2 derivates from can be robustly visualized with TP-Ratio-SO2 in hyperpyretic U251 cells in a ratiometric imaging manner. Two-Photon Bioimaging of Endogenous SO2 in FS-treated Hippocampus. The 13



hippocampus is the primary brain region involved in the generation of FS, and FS can cause neuronal damage and enhance cytogenesis in the hippocampus.43 Taking advantage of its excellent two-photon property, TP-Ratio-SO2 shows predominant tissue penetration and can be used to dual-color monitor the concentration-dependent changes of exogenous SO2 derivatives in liver tissue samples (Figure S10 and Figure S11). Therefore, we proceeded to interrogate its capability for visualizing endogenous SO2 derivates in model FS-treated hippocampus (Figure 6), FS-induced hippocampus injury was modeled according to previously reported methods.44 These FS-treated hippocampus slices were incubated with TP-Ratio-SO2 for 30 min in the absence and presence of HDX, followed by two-photon imaging. As expected, FS treatment triggered elevation of endogenous SO2 derivatives in hippocampus, as indicated by the weaken fluorescence signal in red channel, together with increase of green fluorescence. However, the prominent fluorescence ratio signal change could be efficiently attenuated by L-aspartate-hydroxamate (HDX). These imaging findings demonstrate for the first time that a designed probe enables the ratiometrical visualization of endogenous SO2 fluctuation in rat hippocampus tissues after FS treatment.

CONCLUSION In conclusion, we have designed a TP-FRET-based ratiometric two-photon fluorescence probe for imaging of SO2 derivates produced by febrile seizures in mammal for the first time. This probe, consisted of acedan-anthocyandin dyad, exhibits superior features including significant red-to-green fluorescence change with 14


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large emission shift, fixed two-photon excitation maximum, fast response, high sensitivity, and good selectivity, thereby allowing ratiometrical imaging of SO2 derivates in living cells and deep tissues. What’s more, further imaging studies clearly revealed the elevation of endogenous SO2 derivates in hyperpyretic U251 cells and as well as in rat hippocampus with respect to the febrile seizures, manifesting that the new probe would be a robust molecule tool to understand the role of SO2 in FS-associated neurological diseases. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]; Fax: +86-731-88822523. ACKNOWLEDGMENTS This work was financially supported by NSFC (21505006, 21735001, 21575018), the Foundation for Innovative Research Groups of NSFC (21521063), Hunan Provincial Natural Science Foundation of China (2017JJ3332), the Scientific Research Fund of Hunan Provincial Education Department (16C0033, 17A002). ASSOCIATED CONTENT Supporting Information The supporting information is available free of charge on the ACS Publications website at DOI: Materials and instruments, synthesis of intermediates, experimental details on quantum yield and two-photon measurements and cytotoxicity assay, additional spectroscopic data and imaging patterns, and NMR and mass spectra of 15



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(40) Yang, X. G.; Zhou, Y. B.; Zhang, X. F.; Yang, S.; Chen, Y.; Guo, J. R.; Li, X. X.; Qing, Z. H.; Yang, R. H. A TP-FRET-Based Two-Photon Fluorescent Probe for Ratiometric Visualization of Endogenous Sulfur Dioxide Derivatives in Mitochondria of Living Cells and Tissues. Chem. Commun. 2016, 52, 10289–10292. (41) Luo, L. M.; Chen, S.; Jin, H. F; Tang, C. S.; Du, J. B. Endogenous Generation of Sulfur Dioxide in Rat Tissues. Biochem. Biophys. Res. Commun. 2011, 415, 61–67. (42) Jin, H. F.; Du, S. X.; Zhao, X.; Wei, H. L.; Wang, Y. F.; Liang, Y. F.; Tang, C. S.; Du, J. B. Effects of Endogenous Sulfur Dioxide on Monocrotaline-Induced Pulmonary Hypertension in Rats. Acta Pharmacol. Sin. 2008, 29, 1157–1166. (43) Han, Y.; Yi, W. X.; Qin, J.; Zhao, Y.; Zhang, J.; Chang, X. Z. Carbon Monoxide Offers Neuroprotection from Hippocampal Cell Damage Induced by Recurrent Febrile Seizures Through the PERK-Activated ER Stress Pathway. Neurosci. Lett. 2015, 585, 126–131. (44) Yang, Z. X.; Qin, J. Interaction between Endogenous Nitric Oxide and Carbon Monoxide in the Pathogenesis of Recurrent Febrile Seizures. Biochem. Biophys. Res. Commun. 2004, 315, 349–355.

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Scheme 1. Structure and Response Mechanism of TP-Ratio-SO2 to SO2 Derivatives.

23



Page 24 of 31

Scheme 2. Synthetic procedure of TP-Ratio-SO2. Reaction conditions: a) tert-Butyl bromoacetate,

K2CO3;

b)

4-(diethylamino)salicylaldehyde,

MeSO3H;

4-(N-Boc-amino)piperidine, EDC, HOBt; d) CF3COOH; e) 3, EDC, HOBt.

24


c)

Page 25 of 31

0.60

Na2SO3

a

B 150

TP Fl. Intensity

A

Absorbance

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


0.45 0.30 0.15

b

a

120

Na2SO3

b

90 60

c

30

0.00

0 450 500 550 600 650 700 Wavelength/nm

400 480 560 640 720 Wavelength/nm

Figure 1. (A) UV-vis absorption spectra of 10.0 μM TP-Ratio-SO2 (a) and it response to 2 mM Na2SO3 (b) in 10 mM PBS buffered aqueous DMSO solution (7/3 v/v, pH = 7.4). Inset: color change of TP-Ratio-SO2 after treatment with Na2SO3. (B) Two-photon excitation fluorescence spectra of 1.0 μM TP-Ratio-SO2 in the presence (a) and absence (b) of 400 μM Na2SO3 and 1.0 μM compound 3 (c) in 10 mM PBS buffered aqueous DMSO solution (7/3 v/v, pH = 7.4). λex =760 nm. Inset: fluorescence tint change of TP-Ratio-SO2 after treatment with Na2SO3.

25



B

16

8

B

12

6

8 4

4

S/B

F500/F640

6

A

Fluorescence intensity(10 )

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

Page 26 of 31

2

8 6 4 2 0 0 15 30 45 [Na2SO3]/M

0

0

450 500 550 600 650 700 Wavelength/nm

0

80 160 240 320 400 [Na2SO3]/M

Figure 2. (A) One-photon fluorescence spectral changes of 1.0 μM TP-Ratio-SO2 with various concentrations of Na2SO3 (0−400 μM) in 10 mM PBS buffered aqueous DMSO solution (7/3 v/v, pH = 7.4). λex = 390 nm. (B) the corresponding fluorescence intensity ratio (F500/F640) of TP-Ratio-SO2 as a function of Na2SO3 concentrations (0−400 μM). Inset: linear responses of the signal-to-background ratio (S/B = (F500/F640)n/(F500/F640)0) values of TP-Ratio-SO2 to changing Na2SO3 concentrations (0−50 µM), where the terms of (F500/F640)n and (F500/F640)0 take into account the ratios of donor and acceptor emission intensities of the probe with and without Na2SO3, respectively.

26


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


F500/F640

Page 27 of 31

6.0

200 M

4.5

150M

3.0

Na2SO3 added

80 M 30M

1.5

0M

0.0 0

3

6 9 Time/s

12

Figure 3. Real-time records for fluorescence emission ratio (F500/F640) changes of 1.0 μM TP-Ratio-SO2 in the presence of different concentrations of Na2SO3 (0, 30, 80, 150, 200 µM). λex = 390 nm.

27



Competing species

Competing species + Na2SO3

4.5

500

/F

640

6.0

F

3.0

3 -

-

SO H

N

-

S

O C H

C

2

H

2O H5 2O

P

3

SO

4 2-

SH Ac O N O S 2 2O 2

G

ys

cy

C

H

-

-

Br I -

l C

F

nk

-

0.0

3 -

1.5

bl a

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

Page 28 of 31

Figure 4. Fluorescence ratio (F500/F640) changes of TP-Ratio-SO2 for Na2SO3 in the presence of other common species in 10 mM PBS buffered aqueous DMSO solution (7/3 v/v, pH = 7.4). The concentrations of Na2SO3 and other species were 350 µM and 500 µM, respectively.

28


Page 29 of 31

A

Green Channel

Red Channel

Overlay

Normal

Bright Field

+ HDX

Hyperpyrexia Hyperpyrexia

C

B 1.6

Ratio

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


Normal

Heperpyrexia

1.2

Hyperpyrexia +HDX

0.8

Hyperpyrexia

Normal

0.4

+ HDX

Figure 5. (A) Two-photon fluorescence images of endogenous SO2 derivates of U251 cells using 1.0 μM TP-Ratio-SO2 under different conditions: (upper) normal; (middle) hyperpyrexia; (under) hyperpyrexia pretreated with 100 nM HDX. (B) Histograms of fluorescence pixel intensity ratio in panel (A). (C) Flow cytometry analysis using 1.0 μM TP-Ratio-SO2 under different conditions as (A). The images were collected at 490-520 nm (green channel) and 610-660 nm (red channel) upon excitation at 760 nm. Scale bar: 20 μm. 29



Green Channel

Red Channel

Page 30 of 31

Ratio

Normal

Febrile Seizure

Febrile Seizure +HDX

Figure 6. Two-photon ratiometric fluorescence images of endogenous SO2 derivates in rat hippocampus tissues using 5 μM TP-Ratio-SO2 under different conditions: (upper) normal; (middle) treatment of febrile seizure; (under) treatment of febrile seizure in the presence of HDX. The images were collected at 490-520 nm (green channel) and 610-660 nm (red channel) upon excitation at 760 nm. Scale bar: 50 μm.

30


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For TOC only

31


Visualizing Endogenous Sulfur Dioxide Derivatives in Febrile-Seizure

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