A FRET-based Fluorescent Probe for the Selective Imaging of

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A FRET-based Fluorescent Probe for the Selective Imaging of Hydroxylamine in Living Cells Baoli Dong, Minggang Tian, Xiuqi Kong, Wenhui Song, Yaru Lu, and Weiying Lin Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b02737 • Publication Date (Web): 12 Aug 2019 Downloaded from pubs.acs.org on August 12, 2019

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Analytical Chemistry

A FRET-based Fluorescent Probe for the Selective Imaging of Hydroxylamine in Living Cells Baoli Dong, Minggang Tian, Xiuqi Kong, Wenhui Song, Yaru Lu and Weiying Lin* Institute of Fluorescent Probes for Biological Imaging, School of Chemistry and Chemical Engineering, School of Materials Science and Engineering, University of Jinan, Jinan, Shandong 250022, People’s Republic of China * E-mail: [email protected]. ABSTRACT: Hydroxylamine (HA) is an important product of cell metabolism and plays significant roles in many biological processes, and therefore, real-time imaging of HA is of great importance for the in-depth study of its physiological and pathological functions. However, HA-specific fluorescent probe is currently lacking primarily because the highly selective HAresponsive site is undeveloped. To address this critical issue, we present a HA-specific FRET-based fluorescent probe (RhChr) for the selective detection of HA in living systems. Inspired by aza-Michael addition, the unsaturated system appended with an iminium ion was employed as the new HA-specific response site. In response to HA, RhChr provided ratiometric signal output with excellent selectivity towards HA over biothiols and ammonia. We have demonstrated that RhChr could be applied for the ratiometric imaging of endogenous HA in living cells, and the evaluation of xanthine oxidase (XOD) activity in living organs.

Hydroxylamine (HA) is an important product of cell metabolism and plays significant roles in many biological processes.1-4 HA acts as an important intermediate to participate in the conversion process of L-arginine to nitric oxide (NO) and nitroxyl (HNO), which are the well-known signaling molecules with significant biological effects in living systems.5-9 HA itself also possesses various biological activities.10 For example, HA could dilate resistance arterioles of the blood vessels of rat’s kidney and mesentery as a vasodilator, relax the rat endotheliumdenuded aortic rings with a dose-dependent manner, and inhibit the release of insulin and activate K+ channels.11-15 In addition, HA has also been demonstrated to be an inhibitor for virus and many enzymes in liver and kidney.16 Despite the important biological roles of HA, many aspects of its chemistry in living biological systems still remain unexplored, in part because of the lack of the specific methods for the real-time detection of HA in complex biological environment. Fluorescence imaging is an important technique for the detection of bioactive molecules due to the high sensitivity, nondestructive detection and real-time test.17-22 Very recently, a N-doped carbon dots sensor for the fluorescent detection of hydroxylamine has been developed.23 However, HAspecific fluorescent probes for the imaging of HA in living systems have not been developed yet because the highly selective HA-responsive site is undeveloped. Recently, a turn-on fluorescent probe for the detection of HA was developed.24 However, this probe is also an excellent probe for H2S, and thus it cannot be applied for the selective detection of cellular HA due to the intense interference from cellular thiols.25 As such, attaining high specificity to HA over the other nucleophilic biomolecules is a critical challenge for the design of HA-specific probes.

Herein, we present a highly specific fluorescent probe (RhChr) for the ratiometric imaging of cellular HA, for the first time. Initially, we endeavoured to seek a highly selective HA-responsive site. Inspired by aza-Michael addition of HA to unsaturated system which could proceed rapidly at mild conditions,26-28 we employed iminium ion as the strong electron withdrawing group, and envisioned that the unsaturated system appended with iminium ion could serve as a novel HA-specific response site (Scheme 1A). On the other hand, benzopyrylium derivatives are a sort of excellent dyes due to their prominent optical properties including high fluorescence quantum yield and sufficient photostability.29-31 By embedding the potential HA-specific response site into benzopyrylium dye, we fabricated a HA-responsive dye (Chr) with long emission wavelength. Subsequently, according to our previous works on Förster resonance energy transfer (FRET),32-33 we selected rhodamine B as energy donor and employed Chr as energy acceptor, to develop a new FRET system (RhChr) for the ratiometric detection of HA (Scheme 1B). In response to HA, RhChr could be converted to RhChrHA by aza-Michael addition, resulting in the ratiometric signal output, which could alleviate the interference from variations in probe concentration or excitation intensity relative to turn-on signal output.32 Meanwhile, theoretical calculations demonstrated that the hydrogen bond between the carbonyl of benzoic acid and amine (O…H distance = 2.05 Å; ∠O…H-N = 157.88°) exists in RhChr-HA (Figure S1). This hydrogen bond could exert a stabilizing effect for RhChr-HA, and be beneficial for the reaction between RhChr and HA.

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Analytical Chemistry = 7.2 Hz), 7.99 (s, 1H), 7.86 (t, 1H), 7.78 (m, 4H), 7.58 (m, 2H), 7.36 (s, 1H), 7.25 (m, 1H), 7.16 (m, 7H), 6.95 (s, 2H), 3.64 (m, 20 H), 1.24 (t, 6H), 1.19 (t, 12H). 13C NMR (100 MHz, DMSOd6) δ 167.17, 166.98, 158.51, 158.20, 157.54, 156.16, 155.57, 154.67, 142.49, 139.51, 135.61, 134.79, 133.03, 132.72, 132.17, 131.36, 131.26, 131.03, 130.96, 130.85, 130.75, 130.37, 130.28, 130.21, 129.56, 128.04, 125.65, 124.49, 121.96, 118.92, 118.05, 117.72, 117.58, 117.02, 115.99, 115.96, 114.79, 114.27, 113.53, 107.03, 96.74, 96.39, 46.57, 45.84, 45.58, 12.89. HRMS m/z calcd. for C58H61N5O52+ [M]+ 453.7331, found 453.7366.

(A) Design of HA-specific site based on aza-Michael addition

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Scheme 1. (A) Design of a novel HA-specific site based on azaMichael addition of HA to unsaturated system. (B) Design of HAspecific ratiometric fluorescent probe RhChr.

EXPERIMENTAL SECTION Synthesis of compound Chr. Piperazine (4.30 g, 50 mmol) in 20 mL mixed solvent (H2O: 2-methoxyethanol, v:v= 1:1) was heated at 120 °C for 0.5 h. Then, 1-(4fluorophenyl)ethanone (1.38 g, 10 mmol) in 5 mL 2methoxyethanol was added to the above solution within 1 h at 120 °C. After 4 h, the mixture was poured into 100 mL ice water slowly, and the white solid was precipitated and purified by column chromatography to afford 1-(4(piperazin-1-yl)phenyl)ethanone (1.53 g, 75%). Subequently, 1-(4-(piperazin-1-yl)phenyl)ethanone (612 mg, 3 mmol) and 2-(4-(diethylamino)benzoyl)benzoic acid (891 mg, 3 mmol) were dissolved in 5 mL concentrated sulfuric acid, and this mixture was heated at 90 °C for 4 h. After cooled to room temperature, the mixture was poured into 20 mL ice water slowly, and 5 mL perchloric acid was added dropwise. The mixture was filtrated to afford red solid, which was further purified by column chromatography using DCM and MeOH (V/V, 3:1) as eluent to afford compound Chr perchlorate (1.20 g, 69%). 1H NMR (400 MHz, DMSO-d6) δ 12.23 (s, 1H), 8.82 (s, 1H), 8.34 (d, 2H, J = 8.4 Hz), 8.17 (d, 1H, J = 7.6 Hz), 7.87 (t, 1H), 7.79 (t, 1H), 7.53 (d, 1H, J = 7.2 Hz), 7.34 (s, 1H), 7.21 (d, 3H, J = 9.2 Hz), 7.13 7.21 (d, 1H, J = 8.8 Hz), 3.74 (t, 4H), 3.64 (q, 4H), 3.27 (t, 4H), 3.17 (s, 1H), 1.24 (t, 6H). 13C NMR (100 MHz, DMSO-d6) δ 167.34, 165.93, 164.84, 158.15, 157.98, 154.71, 154.10, 133.25, 130.75, 130.64, 130.46, 129.83, 129.56, 118.78, 114.79, 96.84, 49.07, 45.53, 43.98, 42.98, 12.90. HRMS m/z calcd. for C30H32N3O3+ [M]+ 482.2438, found 482.2450. Synthesis of the probe RhChr. A mixture of rhodamine B (479 mg, 1 mmol) and 1 mL POCl3 in 15 mL C2H4Cl2 was stirred at 60 °C for 4 h. Then, the mixture was evaporated and dissolved in 5 mL CH2Cl2 to form red solution. Subsequently, the red solution was added dropwise to the solution of compound Chr (581 mg, 1 mmol) and 0.2 mL EtN3 in 5 mL CH2Cl2 at 0 °C within 0.5 h, and the mixture was then stirred for 3 h. 0.5 mL perchloric acid was added dropwise to the mixture, and then stirred for 0.5 h. The resulting mixture was evaporated and purified by pre-HPLC to afford product RhChr perchlorate (55.2 mg, 5%). 1H NMR (400 MHz, DMSO-d6) δ 8.34 (d, 2H, J = 10.4 Hz), 8.18 (d, 1H, J

Imaging of HA in living cells. (A) For the imaging of exogenous HA, HepG2 cells were first treated with 5 μM RhChr for 30 min at 37 °C, and washed with PBS (pH 7.4) to remove excess RhChr. Then cells were incubated with 100 μM HA for 30 min. (B) For the imaging of endogenous HA in living cells: RAW 264.7 macrophages were incubated with 20 μg/mL LPS, 50 μg/mL L-Arg, 0.01 μg/mL IFN-γ and 5 μM RhChr for 12 h at 37 °C, and washed three times with PBS (pH 7.4). Images were acquired with Nikon A1R confocal microscope with a 100× objective lens. Imaging of HA in tissues. Tissues were obtained from the mice liver, and treated with 5 μM RhChr in an incubator for 30 min and then washed with PBS (pH 7.4) three times. Subsequently, the tissues were treated with 100 μM HA for 30 min. Images were acquired with Nikon A1R confocal microscope at Z-scan mode with a 10× objective lens. Detection of xanthine oxidase in living organs. After the euthanasia of mice by cervical dislocation, we got different organs (liver, heart, lung, etc) from mice carefully. The organ tissues were dried out by filter papers and weighted. Then, saline solution (0.9% NaCl) was added in accordance with the proportion of 10% (weight/volume). The mixture was ground for 20 min below 0 °C and centrifuged at for 10 min at 4 °C to obtain the supernatants. Subsequently, the saline solution was added into the super natants to prepare 10% organ homogenates for the measurement. For the detection of XOD relative level in different living organs. (1) Control group: A mixture of 0.15 mL HA (1 mM), 0.50 mL RhChr (10 μM in MeOH) and 1.25 mL PBS (pH = 7.4) were mixed, and then its fluorescence was determined at different times. (2) XOD group: 0.50 mL HX (1 mM), 0.50 mL XOD (0.04 U/mL) and 0.25 mL NaCl (0.9%) were mixed and kept at 37 °C for 30 min. Subsequently, 0.15 mL HA (1 mM) was added and kept at room temperature for 10 min. After that, 0.50 mL RhChr (10 μM in MeOH) was added, and the fluorescence of the mixture was determined at different times. (3) Heart, liver, lung, kidney and spleen groups: 0.50 mL HX (1 mM), 0.50 mL PBS (pH = 7.4), 0.10 mL NaCl (0.9%) and 0.15 mL 10% tissue sample from different living organs were mixed and kept at 37 °C for 30 min. Subsequently, 0.15 mL HA (1 mM) was added. After 10 min, 0.50 mL RhChr (10 μM in MeOH) was added, and the fluorescence spectra of the mixture was determined at different times. For the detection of XOD activity in living liver organs. Allopurinol (I) group: 0.50 mL HX (1 mM), 0.40 mL PBS (pH = 7.4), 0.10 mL allopurinol (0.1 mM), 0.10 mL NaCl (0.9%) and 0.15 mL 10% liver tissue sample were mixed and kept at 37 °C for 30 min. Subsequently, 0.15 mL HA (1 mM) was added and kept at room temperature for 10 min. After that, 0.50 mL RhChr (10 μM in MeOH) was added, and the


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fluorescence of the mixture was determined at different times. Allopurinol (II) group: 0.50 mL HX (1 mM), 0.50 mL allopurinol (0.1 mM), 0.10 mL NaCl (0.9%) and 0.15 mL 10% liver tissue sample were mixed and kept at 37 °C for 30 min. Subsequently, 0.15 mL HA (1 mM) was added. After 10 min, 0.50 mL RhChr (10 μM in MeOH) was added, and the fluorescence of the mixture was determined at different times.

RESULTS AND DISCUSSION Spectroscopic response of the probe RhChr towards HA. We initially evaluated the optical properties of RhChr by UV-Vis absorption and fluorescence spectra. The control compounds Rhodamine B and Chr showed main absorption peak at 550 nm and 580 nm, respectively (Figure 1A). Under excitation at 540 nm, they exhibited fluorescence maxima at 580 nm and 637 nm, indicating the desirable overlap between the fluorescence spectrum of Rhodamine B and absorption spectrum of Chr. Meanwhile, the mixture of equal molar Rhodamine B and Chr showed two fluorescence maxima at 580 nm and 637 nm (Figure S2), while RhChr showed a main fluorescence maximum at 637 nm (Figure 1B), suggesting that FRET process occurs at the excitation state of RhChr. Subsequently, the fluorescence response properties of RhChr towards HA were explored in PBS (pH = 7.4, 20% EtOH). After the treatment of RhChr with HA, the fluorescence intensity at 637 nm descended gradually, accompanying by the increase of that at 590 nm (Figure 1B). The fluorescence intensity ratio (I637/I590) showed a desirable linear relation with the HA concentration of 0-80 μM (R2 = 0.9979), and the detection limit was evaluated to be 2.16 μM (Figure 1C). After the reaction of RhChr with 100 μM or 150 μM HA, the time-dependent fluorescence spectra showed that the probe RhChr could respond to HA within 4 min, indicating (B) 2000

Rho. B Abs Chr Abs Rho.B Em Chr Em

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the fast response of RhChr to HA (Figure 1D and Figure S3). Furthermore, the absorption and fluorescence spectra of rhodamine B and Chr in absence and presence of HA suggest that the response of RhChr to HA could be ascribed to Chr moiety instead of rhodamine B moiety (Figure S4-S5). Meanwhile, HRMS data showed that the peak (m/z = 470.2454) corresponding to RhChr-HA (calcd. m/z = 470.2438) could be observed clearly after the reaction of RhChr with HA (Figure S6), indicating that the response of RhChr towards HA is probably on the basis of aza-Michael addition. Taken together, RhChr could potentially act as a highly sensitive fluorescent probe for the detection of HA in living systems.

Subsequently, we assessed the selectivity of RhChr to HA. Under excitation at 540 nm, only HA could trigger obvious changes of the fluorescence intensity ratio (I637/I590), suggesting that RhChr is highly selective for HA over other biologically relevant species (Figure 2A and Figure S7). Given that the strong nucleophilic biomolecules (GSH, H2S, etc) and other reactive sulphur species (e.g. SO2) are ubiquitous in living systems and liable to interfere with the detection of HA, we further conducted the fluorescence titration experiments to confirm the high selectivity of RhChr to HA. As shown in Figure 2B and Figure S8-S10, the fluorescence spectra of RhChr showed no significant changes upon the treatment with GSH (0-10 mM), S2- (0-500 μM) or SO32(0-500 μM). In consideration of the endogenous level of GSH (0-10 mM), H2S (10-160 μM) and SO2 (~ 15 μM),34-36 these sulphur-containing biomolecules exerted minimal disturbance for the detection of HA in biological systems. Time-dependent fluorescent spectra demonstrated that RhChr exhibited negligible interference from GSH, S2- or SO32- for a long time (Figure S11-S13). Furthermore, the fluorescence response of RhChr to amine species (NH3, NH4+ and L-arginine) which are prevalence in living systems and relevant to the metabolism of HA, also demonstrated the remarkable selectivity of the probe over these biological amine derivatives (Figure 2B and Figure S14-S19). In addition, the fluorescence ratio I637/I590 of RhChr displayed faint variation in pH 3.0-9.0 under excitation at 540 nm (Figure S20). After the treatment with HA, RhChr exhibited obvious response to HA under physiological pH condition. Additionally, time-dependent fluorescence spectra of RhChr under ceaseless irradiation clearly showed its desirable photostability (Figure S21). Therefore, RhChr could act as a highly selective probe for the ratiometric imaging of HA in living systems.

Figure 1. (A) Normalized absorption and fluorescence spectra of 5 μM rhodamine B (Rho. B) and Chr in PBS. Gray area represents the overlap between the fluorescence spectrum of Chr and the absorption spectrum of rhodamine B. (B) Fluorescence spectra of 5 μM RhChr in the presence of different concentrations of HA (0-200 μM) in PBS. (C) Fluorescence intensity ratios (I637/I590) of RhChr treated with various HA concentrations. Inset: linear relationship between I637/I590 and HA concentration in the range 0-80 μM. (D)


Analytical Chemistry (A) 3.6

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Figure 2. (A) Fluorescence intensity ratios (I637/I590) of 5 μM RhChr treated with various species in PBS. 1, blank; 2, GSH; 3, Cys; 4, Hcy; 5, SO32-; 6, S2-; 7; Fe2+; 8, Fe3+; 9, Cu2+; 10, Ca2+; 11, Zn2+; 12, Mg2+; 13, Mn2+; 14, I-; 15, O2•−; 16, NO; 17, OCl-; 18, ·OH; 19, H2O2; 20, Glucose; 21, NH3; 22, NH4+; 23, L-Arginine; 24, VC; 25, HA. Concentration: GSH, 1 mM; other analytes, 100 μM. (B) Dose-dependent fluorescence intensity ratios (I637/I590) of 5 μM RhChr treated with various species in PBS. λex = 540 nm.

Ratiometric imaging of HA in living cells and tissues. Next, we explored the capability of RhChr to visualize cellular HA. MTT data showed that RhChr possessed no marked cytotoxicity to HepG2 cervical cancer cells when its concentration was below 20 μM (Figure S22). When the HepG2 cells were incubated with 5 μM RhChr, weak orange fluorescence in Channel 1 and strong red fluorescence in Channel 2 can be observed clearly (Figure 3A). Upon the addition of 100 μM HA, the orange fluorescence enhanced obviously while the red fluorescence reduced (Figure 3A). Furthermore, given that the large negative membrane potential (150-180 mV) across the inner membrane of mitochondria,37 RhChr could be liable to accumulate in mitochondria because of its two cations. Colocalization experiments were then conducted in HepG2 cells using RhChr and a comercial mitochondria-specific dye (MitoTracker) to evaluate the mitochondria-targeting property of RhChr (Figure 3B). The Pearson’s correlation coefficient between green fluorescence from Mito-Tracker and the red fluorescence from RhChr was evaluated to 0.90, suggesting that RhChr mainly distributes in mitochondria. Therefore, RhChr can be applied for the ratiometric imaging of cellular HA. We then evaluated the feasibility of RhChr to image endogenously produced HA in RAW 264.7 macrophages. It has been established that interferon-γ (IFN-γ), lipopolysaccharide (LPS) and L-arginine (L-Arg) could regulate the expression of cellular nitric oxide (NO) synthase and result in the micromolar quantities of nitric oxide production.38-39 Recently, an increasing evidence showed that NO could be convert to nitroxyl (HNO) by the reduction of

Figure 3. (A) Images of HepG2 cells treated with 5 μM RhChr in absence or presence of 100 μM HA. Channel 1, λem = 570-620 nm; Channel 2, λem = 663-738 nm. λex = 561 nm. Scale bar = 10 µm. (B) Colocalization experiment of HepG2 cells treated with MitoTracker Green and RhChr. Mito-Tracker Green channel, λem = 500550 nm, λex = 488 nm; RhChr channel, λem = 663-738 nm, λex = 561 nm. Scale bar = 10 µm.

biological alcohols with reducing capacity, such as ascorbate or tyrosine.40 In the presence of excess reductive thiols (RSH), HNO reacts with RSH to form S-nitrosothiol (RSNHOH) which is subsequently reduced by RSH and releases HA.9 Accordingly, we treated RAW 264.7 macrophages with IFNγ/LPS/L-Arg to obtain a biological model of endogenous HA (Figure 4A). After the stimulation with IFN-γ/LPS/L-Arg for 12 h, the RhChr-treated cells showed decreased fluorescence ratio (IChannel 2/IChannel 1) in comparion with the unstimulated cells (Figure 4B). It suggested that the endogenous HA produced after the stimulation by IFN-γ/LPS/L-Arg and then reacted with RhChr in cells. Therefore, the probe RhChr could be employed for the ratiometric imaging of endogenous HA in living cells. (A)

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Figure 4. (A) Schematic image of the endogenouely produced HA in living cells. (B) Images of endogenous HA in RAW 264.7 macrophages stained with 5 μM RhChr and stimulated without or with 20 μg/mL LPS, 50 μg/mL L-Arg and 0.01 μg/mL IFN-γ for 12 h. Channel 1, λem = 570-620 nm; Channel 2, λem = 663-738 nm. λex = 561 nm. Scale bar = 10 µm. (C) Ratios of fluorescence intensities in Channel 1 and Channel 2 obtained from (B). The error bars represent standard deviation (± S.D.).

The capability of RhChr for the ratiometric imaging of HA was further evaluated using fresh liver tissues from a living mouse. Under excitation at 561 nm, the tissues only stained with 5 μM RhChr showed weak fluorescence (Channel 1)


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from rhodamine B moiety with a penetration depth of 45 μm approximately, and strong fluorescence(Channel 2) from Chr moiety (Figure 5 and Figure S23). However, when the tissues were further treated with 100 μM HA, it can be observed that the fluorescence from rhodamine B moiety increased and the fluorescence from Chr moiety decreased clearly (Figure 5 and Figure S24). Therefore, the probe RhChr can be employed for the imaging of HA in tissues by a ratiometric manner.

reagent, and allow for the accurate detection.46 Taken together, RhChr could serve as a novel ratiometric signal reporter for the evaluation of XOD activity in living organs. .

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Figure 5. Imaging of the liver tissue stained with 5 μM RhChr in the absence (A) or presence (B) of 100 μM HA under excitation at the penetration depth of 10 μm and 20 μm. Channel 1, λem = 570-620 nm; Channel 2, λem = 663-738 nm. λex = 561 nm. Scale bar = 50 μm.

Evaluation of xanthine oxidase (XOD) activity in living organs. Furthermore, the capability of RhChr as a new ratiometric signal reporter to detect xanthine oxidase (XOD) activity in living organs was investigated. XOD is an important enzyme in the catabolism of purines, and its abnormal level is closely associated with many diseases such as gout, inflammation and hypertension.41-42 Evaluation of XOD activity is of great importance for the clinical diagnosis and the discovery of XOD inhibitor in drug development. Hypoxanthine (HX)-XOD is a classic system that commonly used for the production of O2•−, which can be easily scavenged by HA.43 Consequently, XOD activity measurement could be achived using a HA probe. In this work, as a demonstration, we utilized RhChr as a ratiometric signal reporter to determine the level of residual HA after its reaction with O2•− which was generated by enzymatical reaction between HX and XOD from animal tissues, and then detected XOD level and evaluated XOD activity in living organs (Figure 6A and Figure 6B). XOD group demonstrated that a portion of HA could be scavenged by O2•− and it results in larger ratiometric value (I637/I590) in comparison with control group (Figure 6C). When XOD was replaced with different living tissue samples, the corresponding I637/I590 values increased in varying degrees, indicating that certain amount of active XOD exists in various living tissues. According to the experimental results, among these organs, liver showed larger I637/I590 value than the other organs, suggesting that XOD concentration in liver is higher than that in other organs, and it consists with the previous report that XOD is mainly distributed in liver.44 We then employed RhChr to evaluate XOD activity change in living liver tissues by allopurinol, which is a well-known XOD inhibitor that commonly used for the treatment of gout.45 Compared with liver group, the addition of allopurinol lead to obviously lower I637/I590 values, indicating that XOD activity in living liver tissues suffer inhibition from allopurinol. Compared with the turn-on signal output of traditional XOD activity detection methods, the ratiometric signal output described herein, could minimize the interference from variations in the concentration of chromogenic reagent or fluorometric

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Figure 6. (A) Mechanism of the evaluation of XOD activity by RhChr. (B) Experiment processes for the detection of XOD in different organs. (C) Time-dependent fluorescence intensity ratios (I637/I590) of 0.15 mL HA (1 mM) and 0.5 mL RhChr (10 μM in MeOH) in different systems. Control group: 1.25 mL PBS; XOD group: 0.50 mL HX (1 mM) + 0.50 mL XOD (0.04 U/mL) + 0.25 mL NaCl (0.9%); Heart, liver, lung, kidney and spleen groups: 0.50 mL HX (1 mM) + 0.50 mL PBS + 0.10 mL NaCl (0.9%) + 0.15 mL 10% tissue sample; Allopurinol (I) group: 0.50 mL HX (1 mM) + 0.40 mL PBS + 0.1 mL allopurinol (0.1 mM) + 0.10 mL NaCl (0.9%) + 0.15 mL 10% liver sample; Allopurinol (II) group: 0.50 mL HX (1 mM) + 0.50 mL allopurinol (0.1 mM) + 0.10 mL NaCl (0.9%) + 0.15 mL 10% liver sample. λex = 540 nm.

CONCLUSIONS In conclusion, we have developed a unique FRET-based fluorescent probe RhChr for the selective ratiometric imaging of HA. An unsaturated system appended with an iminium ion was employed as the new HA-specific response site. In response to HA, RhChr can provide a rapid ratiometric fluorescence signal with remarkable selectivity over the ubiquitous biothiols and ammonia. RhChr could be applied for the ratiometric imaging of the endogenously produced HA in living cells. Meanwhile, RhChr could act as a ratiometric signal reporter to evaluate XOD activity in living organ. We expect that this probe could be extensively employed for the in-depth study of the physiological and pathological progresses of HA in living systems, and the discovery of new XOD inhibitor in drug development.

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


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

Experimental details, synthesis of the probe, absorption and fluorescence spectra, characterization data.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected].

Notes The authors declare no conflict of interest.

ACKNOWLEDGMENT This work was financially supported by NSFC (21472067, 21672083, 21877048, 51602127), Taishan Scholar Foundation (TS 201511041), and the startup fund of the University of Jinan (309-10004).

REFERENCES (1)

Saransaari, P.; Oja, S.S. Taurine release modified by nitric oxide-generating compounds in the developing and adult mouse hippocampus. Neuroscience 1999, 89, 1103-1111. (2) Ryba, M.S.; Gordon-Krajcer, W.; Walski, M.; Chalimoniuk, M.; Chrapusta, S.J. Hydroxylamine attenuates the effects of simulated subarachnoid hemorrhage in the rat brain and improves neurological outcome. Brain Res. 1999, 850, 225-233. (3) Louters, L.L.; Scripture, J.P.; Kuipers, D.P.; Gunnink, S.M.; Kuiper, B.D.; Alabi, O.D. Hydroxylamine acutely activates glucose uptake in L929 fibroblast cells. Biochimie. 2013, 95, 787-792. (4) Nishigaya, Y.; Fujimoto, Z.; Yamazaki, T. Optimized inhibition assays reveal different inhibitory responses of hydroxylamine oxidoreductases from beta- and gammaproteobacterial ammonium-oxidizing bacteria. Biochem. Biophys. Res. Commun. 2016, 476, 127-133. (5) Calabrese, V.; Mancuso, C.; Calvani, M.; Rizzarelli, E.; Butterfield, D.A.; Stella, A.M.G. Nitric oxide in the central nervous system: Neuroprotection versus neurotoxicity. Nat. Rev. Neurosc. 2007, 8, 766-775. (6) Irvine, J.C.; Ritchie, R.H.; Favaloro, J.L.; Andrews, K.L.; Widdop, R.E.; Kemp-Harper, B.K. Nitroxyl (HNO): the Cinderella of the nitric oxide story. Trends Pharmacol. Sci. 2008, 29, 601-608. (7) Vetrovsky, P.; Stoclet, J.C.; Entlicher, G. Possible mechanism of nitric oxide production from NGhydroxy-l-arginine or hydroxylamine by superoxide ion. Int. J. Biochem. Cell Biol. 1996, 28, 1311-1318. (8) Taira, J.; Misik, V.; Riesz, P. Nitric oxide formation from hydroxylamine by myoglobin and hydrogen peroxide. Biochim. et Biophys. Acta. 1997, 1336, 502-508. (9) Donzelli, S.; Espey, M.G.; Flores-Santana, W.; Switzer, C.H.; Yeh, G.C.; Huang, J.; Stuehr, D.J.; King, S.B.; Miranda, K.M.; Wink, D.A. Generation of nitroxyl by heme protein-mediated peroxidation of hydroxylamine but not N-hydroxy-L-arginine. Free Radical Bio. Med. 2008, 45, 578-584. (10) Correia, N.A.; Oliveira, R.B.; Ballejo, G. Pharmacological profile of nitrergic nerve-, nitric oxide-, nitrosoglutathioneand hydroxylamine-induced relaxations of the rat duodenum. Life Sci. 2000, 68, 709717.

(11) Moore, P.K.; Burrows, L.; Bhardwaj, R. Hydroxylamine dilates resistance blood vessels of the perfused rat kidney and mesentery. J. Pharm. Pharmacol. 1989, 41, 426-429. (12) DeMaster, E.G.; Raij, L.; Stephen, L.A. Weir, E.K. Hydroxylamine is a vasorelaxant and a possible intermediate in the oxidative conversion of L-arginine to nitric oxide. Biochem. Biophys. Res. Commun. 1989, 163, 527-533. (13) Feelisch, M.; Poel, M.; Zamora, R.; Deussen, A.; Moncada, S. Understanding the controversy over the identity of EDRF. Nature 1994, 368, 62-65. (14) Antoine, M.H.; Ouedraogo, R.; Sergooris, J.; Hermann, M.; Herchuelz, A.; Lebrun, P. Hydroxylamine, a nitric oxide donor, inhibits insulin release and activates K+ATP channels. Eur. J. Pharm. 1996,313, 229-235. (15) Mosen, H.; Salehi, A.; Lundquist, I. Nitric oxide, islet acid glucan-1,4-alpha-glucosidase activity and nutrientstimulated insulin secretion. J. Endocrinol. 2000, 165, 293-300. (16) Gross, P. Biologic activity of hydroxylamine: a review. Crit. Rev. Toxicol. 1985, 14, 87-99. (17) Lakowicz, J.R. Principles of Fluorescence Spectroscopy. Springer, 2006, New York. (18) Dong, B.; Song, X.; Kong, X.; Wang, C.; Tang, Y.; Liu, Y.; Lin, W. Simultaneous near-Infrared and two-photon in vivo imaging of H2O2 using a ratiometric fluorescent probe based on the unique oxidative rearrangement of oxonium. Adv. Mater. 2016, 28, 8755-8759. (19) Gong, Q.; Shi, W.; Li, L.; Ma, H. Leucine aminopeptidase may contribute to the intrinsic resistance of cancer cells toward cisplatin as revealed by an ultrasensitive fluorescent probe. Chem. Sci. 2016, 7, 788-792. (20) Jung, H.S.; Verwilst, P.; Kim, W.Y.; Kim, J.S. Fluorescent and colorimetric sensors for the detection of humidity or water content. Chem. Soc. Rev. 2016, 45, 1242-1256. (21) Yu, H.; Xiao, Y.; Jin, L. A lysosome-targetable and twophoton fluorescent probe for monitoring endogenous and exogenous nitric oxide in living cells. J. Am. Chem. Soc. 2012,134, 17486-17489. (22) Chen, W.; Pacheco, A.; Takano, Y.; Day, J.; Hanaoka, K.; Xian, M. A Single fluorescent probe to visualize hydrogen sulfide and hydrogen polysulfides with different fluorescence signals. Angew. Chem. Int. Ed. 2016, 55, 9993-9996. (23) Wu, J.; Dong, Y.; Yang, X.; Yao, C. N-doped carbon dots sensor for selective detection of hydroxylamine hydrochloride. Optical Materials, 2019, 94, 121-129. (24) Sedgwick, A.C.; Chapman, R.S.L.; Gardiner, J.E.; Peacock, L.R.; Kim, G.; Yoon, J.; Bull, S.D.; James, T.D. A bodipy based hydroxylamine sensor. Chem. Commun. 2017, 53, 10441-10443. (25) Qian, Y.; Karpus, J.; Kabil, O.; Zhang, S.Y.; Zhu, H.L.; Banerjee, R.; Zhao, J.;He, C. Selective fluorescent probes for live-cell monitoring of sulphide. Nat. Commun. 2011, 2, 495. (26) Hurd, C.D.; Patterson, J. The addition of hydroxylamine to ω-nitrostyrene, furylnitroethylene and nitroölefins. J. Am. Chem. Soc. 1953, 75, 285-288. (27) Ku, Y.Y.; Patel, R.R.; Roden, B.A.; Sawick, D.P. Synthesis of substituted heterocycles. Simple method for the introduction of the N-hydroxyurea functionality. Tetrahedron Lett. 1994, 35, 6017-6020.


Page 6 of 8

Page 7 of 8 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 (28) Panfil, I.; Bełzecki, C.; Chmielewski, M.; Suwińska, K. Reaction of α, β-unsaturated sugar lactones with formaldoxime. Tetrahedron 1989, 45, 233-238. (29) Pina, F.; Melo, M.J.; Laia, C.A.T.; Parola, A.J.; Lima, J.C. Chemistry and applications of flavylium compounds: a handful of colours. Chem. Soc. Rev. 2012, 41, 869-908. (30) Chen, W.; Fang, Q.; Yang, D.; Zhang, H.; Song, X.; Foley, J. Selective, highly sensitive fluorescent probe for the detection of sulfur dioxide derivatives in aqueous and biological environments. Anal. Chem. 2015, 87, 609-616. (31) Wang, Y.; Yang, X.F.; Zhong, Y.; Gong, X.; Li, Z.; Li, H. Development of a red fluorescent light-up probe for highly selective and sensitive detection of vicinal dithiolcontaining proteins in living cells. Chem. Sci. 2016, 7, 518524. (32) Yuan, L.; Lin, W.; Zheng, K.; Zhu, S. FRET-based smallmolecule fluorescent probes: Rational design and bioimaging applications. Acc. Chem. Res. 2013, 46, 1462-1473. (33) Yuan, L.; Lin, W.; Xie, Y.; Chen, B.; Zhu, S. Single fluorescent probe responds to H2O2, NO, and H2O2/NO with three different sets of fluorescence signals. J. Am. Chem. Soc. 2012, 134,1305-1315. (34) Yue, Y.; Huo, F.; Li, X.; Wen, Y.; Yi, T.; Salamanca , J.; Escobedo, J.O.; Strongin, R.M.; Yin, C. pH-dependent fluorescent probe that can be tuned for cysteine or homocysteine. Org. Lett. 2017, 19, 82-85. (35) Rajalakshmi, K.; Nam, Y.S.; Youg, S.; Selvaraj, M.; Lee, K.B. Biocompatible silica nanoparticles conjugated with azidocoumarin for trace level detection and visualization of endogenous H2S in PC3 cells. Sensor. Actuat. B-Chem. 2018, 259,307-315. (36) Du, S.X.; Jin, H.F.; Bu, D.F..; Zhao, X.; Geng, B.; Tang, C.S.; Du, J.B. Endogenously generated sulfur dioxide and its vasorelaxant effect in rats. Acta Pharmacol. Sin. 2008, 29, 923-930. (37) Murphy, M.P. Targeting lipophilic cations to mitochondria. BBA-bioenergetics 2008, 1777, 1028-1031. (38) McQuade, L.E.; Ma, J.; Lowe, G.; Ghatpande, A.; Gelperin, A.; Lippard, S.J. Visualization of nitric oxide production in the mouse main olfactory bulb by a celltrappable copper(II) fluorescent probe. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 8525-8530. (39) Winyard, P.G.; Ryan, B.; Eggleton, P.; Nissim, A.;Taylor, E.; Lo Faro, M.L.; Burkholz, T.; Szabó-Taylor, KE.; Fox, B.; Viner, N.; Haigh, R.C.; Benjamin, N.; Jones, A.M.; Whiteman, M. Measurement and meaning of markers of reactive species of oxygen, nitrogen and sulfur in healthy human subjects and patients with inflammatory joint disease. Biochem. Soc. Trans. 2011, 39, 1226-1232. (40) Suarez, S.A.; Neuman, N.I.; Munoz, M.; Alvarez, L.; Bikiel, D.E.; Brondino,C.D.; Ivanović-Burmazović, I.; Miljkovic,J.L.j.; Filipovic,M.R.; Martí, M. A.; Doctorovich, F. Nitric oxide is reduced to HNO by proton-coupled nucleophilic attack by ascorbate, tyrosine, and other alcohols. A new route to HNO in biological media? J. Am. Chem. Soc. 2015, 137, 47204727. (41) Harrison, R. Structure and function of xanthine oxidoreductase: where are we now? Free Radic Biol. Med. 2002, 33, 774-797.

(42) Fang, J.; Yin, H.Z.; Liao, L.; Qin, H.B.; Ueda, F.; Uemura, K.; Eguchi, K.; Bharate, G.Y.; Maeda, H. Water soluble PEG-conjugate of xanthine oxidase inhibitor, PEGAHPP micelles, as a novel therapeutic for ROS related inflammatory bowel diseases. J. Control. Release 2016, 223, 188-196. (43) Shirahata, S.; Kabayama, S.; Nakano, M.; Miura, T.; Kusumoto, K.; Gotoh, M.; Hayashi, H.; Otsubo, K.; Morisawa, S.; Katakura, Y. Electrolyzed–reduced water scavenges active oxygen species and protects DNA from oxidative damage. Biochem. Bioph. Res. Co. 1997, 234, 269-274. (44) Wan, Y.; Zou, B.; Zeng, H.; Zhang, L.; Chen, M.; Fu, G. Inhibitory effect of verbascoside on xanthine oxidase activity. Inhibitory effect of verbascoside on xanthine oxidase activity. Int. J. Biol. Macromol. 2016, 93, 609-614. (45) Zhou, M.; Li, S.; Song, L.; Hu, Q.; Liu, W. 4-(2-(4chlorophenyl)-1-((4chlorophenyl)amino)ethyl)benzene-1, 3-diol is a potential agent for gout therapy as a dual inhibitor of XOD and NLRP3. Phytomedicine 2018, 42, 9-17. (46) Chang, L.; Yao, X.Y..; Liu, Q.; Ning, D.; Wang, Q.; Du, X.M.; Ruan, W.J.; Li, Y. MOF based fluorescent assay of xanthine oxidase for rapid inhibitor screening with realtime kinetics monitoring. Talanta 2018, 183, 83-88.



For Table of Contents Only

HO H

O COOH O

O N

N

O

N OH

N

NH2OH

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N

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Imagingcellular HA Imaging NH2OH

N

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N

N

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N O

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Xanthine oxidase activity evaluation

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