Ratiometric Near-Infrared Fluorescent Probe for Synergistic Detection

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Ratiometric near-infrared fluorescent probe for synergistic detection of monoamine oxidase B and its contribution to oxidative stress in cell and mice aging models Rui Wang, Xiaoyue Han, Jin-Mao You, Fabiao Yu, and Lingxin Chen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b05297 • Publication Date (Web): 05 Feb 2018 Downloaded from http://pubs.acs.org on February 6, 2018

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Analytical Chemistry

Ratiometric near-infrared fluorescent probe for synergistic detection of monoamine oxidase B and its contribution to oxidative stress in cell and mice aging models Rui Wang,ab Xiaoyue Han,ab Jinmao You,ab Fabiao Yu*ab and Lingxin Chen*ab a

Key Laboratory of Life-Organic Analysis, Key Laboratory of Pharmaceutical Intermediates and Analysis of Natural Medicine, College of Chemistry and Chemical Engineering, Qufu Normal University, Qufu 273165, China. b Key Laboratory of Coastal Environmental Processes and Ecological Remediation; Research Center for Coastal Environmental Engineering Technology of Shandong Province, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, China. [email protected]; [email protected]. Phone/fax:+86-35-2109130 ABSTRACT: As new biomarkers, monoamine oxidases (MAOs) play important roles in maintaining the homeostasis of biogenic amines via catalyzing the oxidation of biogenic amines to corresponding aldehydes with the generation of reactive oxygen species (ROS). MAOs have two isoforms, MAO-A and MAO-B. MAO-A is considered to be a major factor of neuropsychiatric and depressive disorders. However, MAO-B is thought to be involved in several neurodegenerative diseases. Therefore, to explore their distinct roles in different diseases, the selective detection of MAOs is essential. Herein, two new types of NIR fluorescent probes, MitoCy-NH2 and MitoHCy-NH2, are provided for synergistic imaging of MAO-B and its contribution to oxidative stress in cells and in mice aging models. These probes are composed of three moieties: heptamethine cyanine as fluorophore, propanamide as recognition group, and triphenylphosphonium cation as mitochondrial targeting group. The amine oxidation and β-elimination reaction can lead to obvious fluorescence increase and color changes from green to blue. The probe MitoHCy-NH2 can be used to synergistically detect MAO-B and its contribution to oxidative stress in replicative senescence model. And the probe MitoCy-NH2 can offer ratiometric near-infrared fluorescence for the selective detection of MAO-B in H2O2-induced cell aging model and in mice aging models. The results reveal that there are different MAO-B levels in different age of mice models. MitoCy-NH2 also can evaluate therapeutic effects of pargyline and selegiline in mice models. The desirable analytical behaviors of our probes make they are useful chemical tools for the selective detection of MAO-B and its contribution to oxidative stress in biosystems.

During the process of aging, neurodegeneration is a fundamental pathological change in the brain. There are many hypothesis for aging. For instance, the ‘free radical theory of aging’ highlights the damage of oxidative stress in abnormal metabolic processes.1 Monoamine oxidases (MAOs) are flavin-depend enzymes which catalyze the oxidation of biogenic amines to yield their corresponding aldehydes along with the generation of reactive oxygen species (ROS).2 MAOs play important roles in maintaining the balance of bio-amines. But the abnormal alteration of MAOs will destroy the homoeostasis of bio-amine metabolism.3 Especially, if the overactivation of MAOs occurs in the brain, the increased ROS levels will lead to oxidative stress and finally accelerate the development of neurodegenerative diseases, such as Parkinson’s disease (PD), Alzheimer's disease (AD), and amyotrophic lateral sclerosis (ALS).4,5 MAOs have two isoforms, MAO-A and MAOB. Although they express 70% sequence identity, the two enzymes possess different substrate preference and specificity. The irregular expression of MAO-A is related to the main etiology for neuropsychiatric and depressive disorders, while the aberrant expression of MAO-B is closely involved into neurodegenerative diseases.1-5 MAO-B mainly localizes in brain and acts on phenethylamine and methylhistamine.2 Compared with old individuals, the levels of MAO-B are higher than those of young ones.6 Therefore, to advance the specific detection of MAO-B can facilitate the discrimination

of its biofunction in complicated biosystems.7,8 However, we consider that if one achieves a goal that the synergistic detection of MAO-B and its catalytic production of ROS can better elucidate the relationship between MAO-B and aging. To date, the methods for MAOs detection mainly include spectrophotometry, radioactivity, enzyme-linked immunoassay, and fluorescence analysis.9-12 Compared with other biodetection technologies, fluorescence probes combined with confocal imaging techniques have now become powerful supporting tools for biological research, because fluorescence imaging offers the promise of accurate detection through noninvasive, real-time, and high-resolution imaging.13-16 Therefore, fluorescence probes can be employed to precisely evaluate enzyme levels while maintaining the enzyme activities in enzyme assays.17-20 Given that MAOs play vital roles in physiological and pathological processes, some efforts have been made to develop fluorescent probes for the detection of MAOs in living biosystems.21-35 The current interests mainly focus on the detection of overall MAOs21-27 or the isomers MAO-A,2831 and only a few fluorescent probes for the selective detection of MAO-B.32-35 However, the MAO-enzymatic reaction involves a potential oxidative stress process. There no fluorescence probe has been developed for the synergistic detection of MAO-B and its contribution to oxidative stress as far as we concerned. It is worth noting that the developed probes for

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Scheme 1 Molecular structures and the proposed detection mechanism of MitoHCy-NH2 and MitoCy-NH2.

a) The synergistic detection mechanism of MitoHCy-NH2 towards MAO-B and reactive oxygen species (ROS). b) The detection mechanism of MitoCyNH2 towards MAO-B.

MAO-B detection with short excitation and/or emission wavelength suffer from short penetration and light scattering.32-35 While the near-infrared (NIR) absorption and emission profiles can increase tissue penetration and minimize the absorbance of heme, water, as well as lipids.13-16 Besides, ratiometric probes that provide the fluorescence ratio from two different fluorescence collection windows can avoid interferences such as uneven loading or the inhomogeneous distribution.36,37 To meet the above challenges, we reported two new NIR ratiometric fluorescent probes, MitoHCy-NH2 and MitoCy-NH2, for the synergistic imaging of MAO-B and its contribution to oxidative stress in catalytic process (Scheme 1). MitoHCyNH2 could offer fluorescence response to the synergistic detection of MAO-B and its contribution to oxidative stress in cell aging models. And the probe MitoCy-NH2 exhibited colorimetric and ratiometric fluorescence response to MAO-B. Both of the two probes could locate in mitochondria due to the mitochondrial targeting group triphenylphosphine cation.38-40 The experimental results demonstrated that the concentration changes of MAO-B in mice were depended on the degrees of aging. The probes could be used as potential chemical tools for the diagnosis of neurodegenerative diseases.

Experimental Section The establishment of H2O2-induced cell aging models. For the establishment of H2O2 induced cell aging models,41 HepG2 cells were plated on 25-Petri dishes. The cells washed by PBS for three times and then added trypsin to digest the cells and divided equally to four groups. When the cells covered with seventy percent of the bottom of the whole dish, we started to treat differently to each groups of cells with H2O2. Group a: the cells maintained in DMEM for 3 h and then in 550 µM H2O2 solution stimulated for 1 h. Group b: the cells maintained in DMEM for 2 h and then in 550 µM H2O2 solution stimulated for 2 h. Group c: the cells maintained in DMEM for 1 h and then in 550 µM H2O2 solution stimulated for 3 h. Group d: the cells maintained in 550 µM H2O2 solution stimulated for 4 h. Then these cells were incubated for 24 h at 37 °C under a humidified atmosphere containing 5% CO2 before imaging. The establishment of replicative ageing models. For the establishment of replicative ageing models,42 cells were plated on 25-Petri dishes. The cells washed by PBS for three times

and then added trypsin to digest the cells. The medium was changed every 48 h. After trypsinization, the cells were passaged. Before replanting, the harvested cells were counted using a cell counter to obtain the number of population doublings (PD). Finally the cumulative population doublings (CPD) of group a – d were 3, 15, 30, and 50, respectively. Western blot analysis. The cells were lysed. Protein extracts were prepared by suspending the cells in 200 µL RIPA lysis buffer containing 1% PMSF (Solarbio, China) and 20 % PhosSTOP (Roche, Germany). The protein extract was stored in a -80 oC refrigerator. Then the protein extracts were quantified with BCA protein assay kit (Biogot, China). And then the equal amounts of protein were electrophoresed on 10% SDSpolyacrylamide gels (Bio-Rad, USA) and transferred to PVDF membranes. The PVDF membrane was incubated with 5% skim milk with gentle shake and then incubated with MAO-B primary antibodies overnight at 4 oC. A horseradish peroxidase (HRP)-conjugated secondary antibody (Cell Signaling Technology, USA) was incubated for 1 h in dark and signals were detected with an enhanced chemiluminescence (ECL) detection system. The results were analyzed by ImageJ software to acquire the grey values. Mice tissue imaging: The tissues used in our imaging experiments were fresh mice brain. Brains were surgically removed from the mouse head and immediately transferred into ice-artificial cerebrospinal fluid (ACSF: 138.6mM NaCl, 3.5 mM KCl, 21 mM NaHCO3, 0.6 mM NaH2PO4, 10 mM Dglucose, 1 mM CaCl2 and 3 mM MgCl2). The pH of the ACSF was 7.4 after saturated with 95% O2/5% CO2. The slices, which contain Substantia nigra, were cut with 300-400 µm at low speed (3 µm/s) and vibration frequency of 70 Hz using a vibrating blade microtome in ACSF. The thick transverse slices in ice cold oxygenated ACSF. After transferred these slices into a storage chamber, the slices rest at RT for at least 1 h prior to staining. Synthesis. Compounds were synthesized according to the general procedure in Scheme S1, and the syntheses details and characterization were described in the Supporting Information. Synthesis of MitoCy-NH2 (6). Mito-Cy (5)43 (0.10 g, 0.1 mmol) and triphosgene (12.0 mg, 0.04 mmol) were in dissolved in 5.0 mL anhydrous CH2Cl2 under Ar condition at 0 °C for 3 min. Then triethylamine (12.1 mg, 0.12 mmol, dissolved in 2.0 mL CH2Cl2) were added to the solution by drop-


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Analytical Chemistry wise. The color of the solution changed into green from blue. 30 min later, the reaction was quenched with saturated aqueous NaHCO3. After removed the solvent in vacuum, the obtained residue was dissolved in 50 mL anhydrous CH2Cl2. Then compound 2 (52.5 mg, 0.3 mmol) and DMAP (20 mg) were added into the solution. Triethylamine (30.3 mg, 0.3 mmol, dissolved in 2.0 mL CH2Cl2) was dropwise into the mixture at 0 °C. The reaction mixture was further stirred at 25 °C, TLC monitored the reaction until the starting material was completely consumed. The mixture was washed three times by saturated NaCl solution, and extracted using CH2Cl2 (50 mL × 3). And removed the organic phase in vacuum, the residue solid was dissolved in 1.5 mL CH2Cl2. 0.5 mL CH2Cl2 containing trifluoroacetate acid (100 uL) was added dropwisely and stirred at room temperature for 30 min. Then the mixture washed three times with saturated NaCl solution, and extracted using CH2Cl2 (50 mL × 3). After removed the organic phase in vacuum, the obtained green solid residue was purified through a silica gel chromatography (200 - 300 mesh) with gradient eluent of ethyl acetate and CH3OH (100:0 to 75:25 v/v) to obtain the probe MitoCy-NH2 (6) as green green solid (27.1 mg, 25%). 1H NMR (500 MHz, CDCl3-D1) δ (ppm): 8.28-8.27 (m, 1H), 8.16-8.14 (m, 1H), 7.98-7.59 (m, 21H), 7.46-7.36 (m, 3H), 7.17-7.16 (m, 1H), 7.07-7.05 (m, 1H), 6.41-6.38 (d, 1H), 6.35-6.32 (d, 1H), 5.51 (s, 1H), 5.15 (s, 1H), 4.57-4.55 (m, 2H), 4.28-4.22 (m, 4H), 3.61-3.59 (t, 2H), 3.52-3.45 (m, 6H), 3.28-3.24 (m, 2H), 3.13-2.99 (m, 4H), 2.05 (s, 2H), 1.87-1.81 (m, 2H), 1.80-1.76 (m, 10H), 1.45-1.29 (m, 12H). 13C NMR (125 MHz, CDCl3-D1) δ (ppm): 171.27, 162.47, 150.03, 143.54, 141.48, 135.10, 135.05, 133.73, 133.65, 130.61, 130.51, 129.16, 128.82, 128.28, 128.25, 127.87, 126.01, 125.96, 124.81, 122.41, 118.41, 118.40, 117.72, 117.71, 115.56, 101.51, 90.85, 65.40, 60.39, 49.52, 48.55, 39.90, 39.71, 39.62, 30.54, 29.67, 29.55, 23.26, 19.21, 14.20, 12.61. LC-MS (ESI+): m/z C69H78N7O3P2+ calcd. 1083.5893, found [M]2+ 541.7940. Synthesis of MitoHCy-NH2 (7): MitoCy-NH2 (6) (0.3 g, 0.25mmol) was dissolved in 10 mL ethanol under Ar condition. The 1 mL NaBH4 aqueous solution (1.5 equiv.) was dropwisely added into the reaction system at 0 °C. The mixture changed its color from green to yellow within 5 min. After added 50 mL CH2Cl2, the mixture was washed with saturated KI solution (100 mL × 3, bubbled with Ar to remove oxygen). After evaporated the solvent by rotary evaporator, the residues were purified by silica chromatography eluted with CH2Cl2 to give a brown solid (0.24g, 82%). 1H NMR (500 MHz, CD3OD1)δ(ppm): 7.78-7.73 (m, 16H), 7.70-7.51 (m, 8H), 7.32-7.29 (m, 1H), 7.26-7.23 (m, 1H), 7.09-7.03 (m, 2H), 6.47-6.44 (m, 2H), 6.36-6.33 (m, 2H), 5.39-5.36 (m, 1H), 5.12 (s, 1H), 4.50-4.45 (t, 2H), 4.19-4.15 (m, 4H), 3.76-3.73 (t, 2H), 3.38 (s, 3H), 3.27-3.24 (m, 2H), 3.18-3.09 (m, 2H), 3.053.00 (m, 2H), 2.81-2.72 (m, 4H), 1.98 (s, 2H), 1.83-1.73 (m, 12H), 1.47-1.30 (m, 12H). 13C NMR (125 MHz, CD3OD) δ(ppm): 159.86, 153.01, 151.10, 148.32, 141.03, 140.01, 137.23, 135.25, 133.89, 130.02, 129.96, 172.93, 127.87, 127.50, 125.20, 123.03, 121.08, 121.62, 119.58, 118.67, 117.86,116.98, 114.95, 112.33, 111.20, 109.15,108.65, 95.03, 77.12, 60.01, 50.03, 49.06, 48.55, 47.01, 38.93, 38.90, 37.75, 36.63, 33.44, 30.07, 30.06, 29.35, 28.20, 23.26, 20.12, 14.49, 13.11. LC-MS (API-ES): m/z C69H79N7O3P+ [M]+ Calcd: 1084.5977, found: 1084.5981.

Results and discussion Design strategy of probes. We now scan three essential factors to initiate the design of new fluorescent probes for the synergistic detection of MAO-B and ROS in biosystems. i) Intracellular distribution and biofunctions of MAO-B: MAO-B locates at the outer membrane of mitochondria. It catalyzes the oxidation of biogenic amines to corresponding aldehydes by generating of ROS (mainly H2O2) as byproduct.7,32-35 However, during this process, superoxide anion (O2•−) may be first produced because O2•− is the initial ROS in biosysytems.44,45 And O2•− can be rapidly converted into other ROS enzymatically or non-enzymically. Follow the origin of ROS in cells, we use the concentration of O2•− to reflect the levels of overall ROS. Therefore O2•− has been selected as the represent of ROS. In this work, we want to investigate the oxidative stress accelerated by MAO-B in the catalysis of monoamines in living cells and in vivo. ii) The choice of specific recognition moieties: propylamine can behave an efficacious response manner towards MAO-B in the molecular structure of the new probe.28-35 And we plan to employ hydrogen abstraction reaction for the detection of O2•−.43,46-48 iii) The selectivity of fluorophore and detection mechanism: heptamethine cyanine has been identified as the fluorophore due to its NIR spectral property and facilitative chemical modification.49-51 The modulation of different substituent groups at the meso-position of cyanine will result in internal charge transfer (ICT)-induced blue or red shifts in ratiometric fluorescent spectra.49-51 Moreover, the iminium cations of cyanine platform can be reduced to hydrocyanine for the further O2•− detection. In the overall design strategy, it is huge challenge for the exact interpretation why the probes show better selectivity toward analytes.13-16 The selectivity of probe towards MAO-B over MAO-A may be result from the steric hindrance of the chemical structure of the probe molecule towards the active center of the enzyme.31 Moreover, the selectivity is based on the reaction kinetics between MAO-B and the fine-tuning MAO-reactive propylamine group.28,32,33As illustrated in Scheme 1, the integration of propylamine into cyanine dye via a carbonate bridge led to spectral red shift. And the reduction destroyed the π-electron system of the polymethine chain and quenched the fluorescence. The introduction of triphenylphosphonium cation could improve the mitochondrial targeting capability of the probe. Finally, we proposed two ratiometric fluorescence probes MitoHCy-NH2 and MitoCy-NH2 for the synergistic detection of MAO-B and O2•− in cell and mice aging models. The detection mechanism was displayed in Scheme 1 and Figure S2. Upon reacted with MAO-B, the fluorophore was released through oxidative deamination, β-elimination, and then selfimmolative discharge of CO2. But the fluorescence recovery of the probe MitoHCy-NH2 required the participation of ROS which offered by enzyme catalysis process. Spectral properties and selectivity. The spectroscopic properties of the probe MitoCy-NH2 towards MAO-B was tested under simulate physiological conditions (10 mM HEPES, pH 7.4, 37 oC). As shown in Figure 1a, the addition of recombinant human MAO-B shifted the maximum of the absorption wavelength from 791 nm to 610 nm along with a color change from green to blue, which indicated that MitoCyNH2 could serve as a “naked eye” colormetric indicator for MAO-B detection. The fluorescence spectra also remarkably exhibited a decrease at 803 nm (ε803 nm = 4.6 × 104 M−1 cm−1, Φ = 0.062) to an increase at 750 nm (ε750 nm = 4.2 × 104 M−1



cm−1, Φ = 0.15). This spectral property made the probe MitoCy-NH2 a good candidate for ratiometric MAO-B detection (Figure 1b and 1c). The logarithm of the ratio (F750 nm/F803 nm) displayed a good linear relationship (r = 0.9911) with the addition of the MAO-B (Figure 1d). The result indicated that MitoCy-NH2 could be used for the ratiometric detection of MAOB. The kinetic behavior is crucial for the application of activity-based fluorescent probe.7,8 We next assessed the enzymatic activity of the recombinant human MAO-B. We obtained the corresponding Michaelis–Menten constants (KM = 10.13 ± 0.28 µM, Vmax = 3.55 nmol mg-1 min-1) (Figure 1e). The value of the KM displayed high affinity between MitoCy-NH2 and MAO-B,21,24 that was, the probe should be a good substrate for MAO-B. MitoCy-NH2 exhibited ideal kinetic behaviors with the maximum ratio fluorescence within 100 min (Figure 1f). In order to confirm the capability of MitoHCy-NH2 for the synergistic detection of MAO-B and its contribution to oxidative stress, we first triggered the ROS-switch with O2•−, and then added MAO-B for the further detection. As shown in Figure S3, MitoHCy-NH2 could successively response to O2•− and MAO-B.

Figure 1. Spectral properties and enzymatic properties of MitoCy-NH2. a) Dose-dependent absorbance spectra of MitoCy-NH2 (10 µM) towards MAO-B. Dates were collected after 2 h incubated with MAO-B at different concentrations (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 µg mL-1) at 37 oC in HEPES (10 mM HEPES, pH 7.4). Dose-dependent emission spectra of MitoCy-NH2 (10 µM) toward MAO-B. b) λex = 730 nm, λem = 770 – 810 nm; c) λex = 650 nm, λem = 700 – 740 nm. All the above experimental conditions were the same as the methods in a); d) The linear fitting curve between the lg(F 750 nm/F 803 nm) and MAO-B. Insert: Intensity ratio of MitoCy-NH2 change with different concentrations of MAO-B; e) Kinetic studies of MAO-B and MitoCy-NH2 over the concentrations range 0 – 100 µM with MAO-B (10 µg mL-1) at 37 oC. Insert: fitted Linewearver- Burk plot. f) Time-depended intensity ratio of MitoCy-NH2 (5 µM) with MAO-B (10 µg mL-1) over 3 h.

In order to verify the selectivity of MitoCy-NH2 towards MAO-B, we evaluated the interferences of various potential intracellular species including ions (Na+, K+, Ca2+, Mg2+, Fe2+, and Zn2+), other reactive species (glucose, vitamin C, arginine, serine, glutathione, and urea), as well as enzymes (MMP-2, MMP-2, MMP-9, MMP-12, phosphate hydrolase, and MAOA). The probe MitoCy-NH2 exhibited a high selectivity towards MAO-B against other potential intracellular species (Figure 2a). We performed the inhibitor experiments to further confirm the fluorescence was triggered by MAO-B. Pargyline (PA) is a specific inhibitor for MAO-B and clorgyline (CL) was a specific inhibitor for MAO-A.3 Obviously, The fluorescence of the probe MitoCy-NH2 was only suppressed by PA (Figure 2b). These result indicated that the fluorescence was surely induced by MAO-B. Then, we tested the selectivity of MitoHCy-NH2 towards various ROS. The result displayed

Figure 2. The selectivities of MitoCy-NH2 towards MAO-B and MitoHCy-NH2 towards ROS. All tests were in HEPES (10 mM HEPES, pH 7.4, 37 oC) and the reaction times of MitoCy-NH2 and MitoHCy-NH2 were 100 and 15 min, respectively. a) MitoCy-NH2 (10 µM ) incubated with the following species: 1, blank；2, Na+(1 mM); 3, K+(1 mM); 4, Ca2+ (1 mM); 5, Mg2+ (1 mM); 6, Zn2+ (1 mM); 7, glucose (10 mM); 8, vitamin C (1 mM); 9, arginine (1mM); 10, serine (1 mM); 11, glutathione (1 mM); 12, urea (20 mM); 13, MMP-2 (100 U/L); 14, MMP-2 (100 U/L); 15, MMP-9 (100 U/L); 16, MMP-12 (100 U/L); 17, phosphate hydrolase (0.5 U/mL); 18, MAO-A (10 µg/mL); 19, MAO-B (10 µg/mL); b) The normalized fluorescence intensity after incubated MitoCy-NH2 (10 µM) with MAO-A (10 µg/mL) and MAO-B (10 µg/mL), respectively. The inhibitors CL and PA (10 µM) were perincubated for 1 h; c) MitoHCy-NH2 (10 µM) incubated with the following species: 1, blank; 2, H2O2 (100 µM); 3, ONOO− (100 µM); 4, NO (25 µM); 5, methyl linoleate hydroperoxide (200 µM); 6, cumene hydroperoxide (200 µM); 7, tert-butyl hydroperoxide (200 µM); 8, ClO− (400 µM); 9, O2•− (25 µM).

Figure 3. Mitochondrial colocalization with MitoHCy-NH2, Rhodamine 123, and Hoechst 33342 in HepG2 cells. a) Cells were treated with 5 µM MitoHCyNH2 for 100 min, b) 1 µg mL-1 Rhodamine 123 for 15 min, and c) 1 µg mL-1 Hoechst 33342 for 30 min. Fluorescence collection windows for red channel a): 700 - 740 nm (λex = 650 nm), green channel b): 550 - 600 nm (λex = 515 nm), and blue channel c): 420 - 480 nm (λex = 405 nm). d) Merged fluorescent images with three channels a) – c). e) Intensity correlated plot between red and green channels. (f) Intensity profile of the red arrow in d) across cell. g) Intensity correlated plot between blue and red channels. h) Intensity correlated plot between blue and green channels. i) Bright-field image of Figure 3.

that MitoHCy-NH2 could provide selective fluorescent response towards O2•− over other ROS including H2O2, ONOO−, NO, methyllinoleate hydroperoxide, cumene hydroperoxide, tert-butylhydroperoxide, ClO− (Figure 2c). These results demonstrated that our probes MitoCy-NH2 and MitoHCyNH2exhibited ideal kinetic behaviors and selectivity, which prompted us to apply the two new probes for detecting the roles of MAO-B in complex biological samples. Mitochondria localization. We now used the probe MitoHCy-NH2 to verify mitochondrial location capability in HepG2 cells (overexpression of MAO-B).31,32 The costaining assay employed a commercial Rhodamine 123 as the mito-


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Analytical Chemistry chondrial dye and Hoechst 33342 as the nucleus fluorescent marker. Fluorescent images in Figure 3 were taken utilizing laser scanning confocal microscopy. The cells were treated with the probe for 100 min, Rhodamine 123 for 15 min, and Hoechst 33342 for 30 min, then were washed with Dulbecco’s Modified Eagle Medium (DMEM) for 3 times before imaging. After reacted with MAO-B, the fluorescence imaging was shown in Figure 3a. We hypothesized that the detection reaction occurred during the transmembrane process. The result showed that the probe MitoHCy-NH2 could be used to synergistic detection of MAO-B and O2•−. The merged image with red, green and blue channels was shown in Figure 3d. The intensity distribution of MitoHCy-NH2 and rhodamine 123 exhibited high correlated plot (Figure 3e) with a high Pesrsons’s coefficient Rr = 0.991. The intensity profile of the linear region of interest across HepG 2 cells (red arrow in Figure 3d) also provided a total synchrony between the two channels. As for the other channels, both the intensity distribution of MitoHCy-NH2 and Hoechst 33342, Rhodamine123 and Hoechst 33342 exhibited low correlated plot (Figure 3g and 3h). As expected, MitoHCy-NH2 could synergistically detect MAO-B/O2•− and selectively accumulate in mitochondria. Selectivity examination in cells. In order to confirm the intracellular fluorescence response resulting from MAO-B, we further performed inhibitor experiments using another probe MitoCy-NH2 due to its capability of ratiometric fluorescence. The SMMC7721 cells in Figure 4a were divided into four groups. The cells in group a were incubated with the probe MitoCy-NH2 for 100 min as control. Prior to incubated with MitoCy-NH2 as described in group a, the cells in group b and c were pretreated with 10 µM PA and CL for 120 min, respectively. The cells in group d were co-pretreated with the two inhibitors for 120 min and then incubated with the probe for 100 min. Then the cells were washed with fresh DMEM 3 times for imaging. The ratiometric fluorescence images was reconstructed with Image-Pro Plus software from two collected windows, channel 1: 770 - 810 nm (λex = 730 nm); channel 2: 700 - 740nm (λex = 650 nm). The imaging results were paralleled in Figure 4a, and the ratio values of Figure 4a were displayed in Figure 4c. The ratio in group a was 0.250 ± 0.008. The cells pretreated with PA produced a remarkable ratio decrease with the value as 0.036 ± 0.002. However, the addition of inhibitor CL could not induce large change in ratiometric fluorescence with a ratio value of 0.190 ± 0.008. The ratio of the cells in group d offered the lowest ratio of 0.027 ± 0.005. All the results of laser scanning confocal microscope images were further verified using flow cytometry analysis (Figure 4b). The results from the two detection methods were highly consistent with each other. We next performed western blot analysis to examine the level changes of MAO-B protein (Figure 4d). It was obvious that the inhibitor PA could limit the level of MAO-B. These consequences revealed that the intracellular fluorescence changes were owing to the activity changes of MAO-B. Fluorescence imaging of MAO-B levels in H2O2 induced and replicative cell aging models. We next investigated the

level changes of MAO-B in two types of cell aging models. HepG2 cells were induced with 550 µM dosage of H2O2 to establish the first aging model.41 As shown in Figure 5a, the

Figure 4. Ratiometric fluorescence images for the selectivity of MitoCy-NH2 in SMMC7721 cells. Two fluorescence collection windows, channel 1:750 – 800 nm (λex = 730 nm); channel 2: 700 – 740 nm (λex = 650 nm). All the cells were washed three times with fresh DMEM before imaging a) Group a: incubated with 5 µM MitoCy-NH2 for 100 min as control. Group b: incubated with 10 µM PA for 120 min then treated as described in group a. Group c: incubated with 10 µM CL for 120 min then treated as described in group a. Group d: incubated with 10 µM PA and 10 µM CL for 120 min then treated as described in group a; b) Flow cytometry analysis of the cells in a); c) The average ratio values in a); d) The average ratio values b).; e) Western blotting analysis of MAO-B in a); f) Quantitative analysis of the levels of active MAO-B. Cell statistics n = 7.

cell models were established at four stages. Group a - d was stimulated for 1 h, 2 h, 3 h, and 4 h, respectively. All the cells were incubated 5 µM MitoCy-NH2 for 100 min, and the cells were washed with fresh DMEM for three times before imaging. As shown in Figure 5b and 5e, the ratio intensities of the four groups were ordered as d > c > b > a. The results of flow cytometry analysis in Figure 5c were consistent well with Figure 5b. It was evident that the levels of MAO-B increased in H2O2 induced aging models. The degree of cellular senescence aggravated with the prolongation of H2O2 stimulation time. The results also indicated that the probe MitoCy-NH2 could be applied as a useful tool to detect the changes of MAO-B in cells. The cell apoptosis were further measured via Jaggregate-forming lipophilic cation 5,5’,6,6’-tetrachloro1,1’,3,3’-tetraethyl benzimidazolylcarbo-cyanine iodide (JC-1) using flow cytometry. The ratio of red/green fluorescence suggested the apoptosis degrees in group a - d as d > c > b > a (Figure 5d and 5f). The levels of MAO-B were evaluated by western blotting assay (Figure 5g and 5h). The levels of MAO-B increased along with the stimulation time of H2O2. The degrees of apoptosis were positive correlation with the levels of MAO-B. The ratiometric fluorescence could directly reflect the degrees of cell apoptosis during H2O2 induced aging process. The results revealed that our probe MitoCy-NH2 could be used as a potential tool to detect MAO-B concentration changes.



Figure 5 Synergistic imaging of MAO-B and O2•− in two types of cell ageing models. a) The protocol of H2O2 induced cell aging models: cells were divided into four groups and incubated with 550 µM H2O2 for 60 min, 120 min, 180 min, and 240 min, respectively. Then the cells were cultured for more two days; b) Fluorescent images with 5 µM MitoCy-NH2 for 100 min; Two fluorescence collection windows, channel 1:750 – 800 nm ( λex = 730 nm); channel 2: 700 – 740 nm (λex = 650 nm). c) Flow cytometry analysis of the cells in b); d) Cell apoptosis analysis using JC-1 by analyzing mitochondrial membrane potentia in b); e) The average ratio values of ratiometric images in b); f) Statistical analysis of JC-1 in b). n = 5; g) Western blotting analysis of MAO-B in b); h ) Quantitative analysis of active MAO-B levels. n = 3. i) Replicative senescence cell aging models: cells were divided into 4 groups. Group a – d: the CPD of each group was 3, 15, 30, and 50. Fluorescent images with 5 µM MitoCy-NH2 at two time points: 10 min and 100 min. All the cells were incubated with5 µM MitoHCy-NH2 for 100 min, then washed with DMEM to remove the redundant probe. j) Flow cytometry analysis of the cells in i); k) Apoptosis analysis by Annexin V/ 7-AAD: (Q1) necrosis (AnnexinV-/7-AAD+), (Q2) late apoptosis (AnnexinV+/7-AAD+), (Q3) viable cells (AnnexinV-/7-AAD-), (Q4) early apoptosis (AnnexinV+/7-AAD-); l) The average ratio values in i); m) Apoptosis analysis of k); n) Western blotting analysis of MAO-B in i); o) Quantitative analysis of the levels of active MAO-B. n = 3.

Replicative senescence is a permanent non-dividing state, which is one of the mechanisms involved in physiological aging.42 We sequentially established the second cell aging model using HepG2 cells. We further employed the probe MitoHCy-NH2 not only to explore the activity of MAO-B but also to detect the by-product ROS in replicative aging models. Cells were cultured and serially passaged until they reached aging. The number of population doublings (PD) was calculated via the formula: PD = (ln[number of cells harvested] – ln[number of cells seeded])/ln2. The cumulative population doublings (CPD) of replicative aging models were determined as 3, 15, 30, 50. And they were allocated as group a, group b, group c, and group d, respectively. We performed an additional assay to validate the response of MitoHCy-NH2 towards O2•− in cells (Figure S6). The results confirmed that O2•− could be generated as byproduct during the enzymatic process. As illustrated in Figure 5i, the four groups were treated with MitoHCy-NH2. Then two time points at 10 and 100 min were selected for acquiring fluorescent images. At the time point of 10 min, the channel 1 of group a – d provided increasing fluorescent images, while the channel 2 was almost no fluorescence emission due to the short reaction time (Figure 5i). The phenomena were attributed to that our probe had detected the outbreak of ROS which depended on the severity of the cell aging models. However, when collected the fluorescent images from the two channels at 100 min, we obtained much stronger images than those at 10 min. Despite the interference of endogenous ROS (Figure S6f), we still attempted to construct ratiometric images to qualitatively measure the level changes of MAO-B and its byproduct in these aging models. The ratio of intensities was ordered as d > c > b > a (Figure 5l). Flow cytometry analysis results (Figure 5j) offered the same trend with Figure 5i. To access the degree of apoptosis during aging process, we performed Annexin V/7-AAD Apop-

tosis Detection Kit for checking the percentage taken by early apoptotic, late apoptotic, and necrotic cells. The apoptosis of the four groups were increased, which was ordered as d > c > b > a (Figure 5k and 5m). The levels of MAO-B at different CPD were explored using western blotting assays (Figure 5n and 5o). The levels of MAO-B increased with the increasing of CPD values. The above results demonstrated that the concentrations of MAO-B increased with the degree of aging. During the enzymatic reaction, the produced ROS might aggravate cell apoptosis process. Fluorescence imaging of MAO-B levels in mice aging models. Our NIR probe would facilitate imaging of the enzymatic process in vivo. The BALB/c mice models were divided into group a - d according to the age: 1, 6, 12, and 24-monthold. All the BALB/c mice were treated with intracranial injection of MitoCy-NH2. The in vivo fluorescent images were obtained at 30 min (Figure 6a). The isolated organs including heart, liver, spleen, and kidney were non-fluorescence, while brain exhibited strong fluorescence in channel 2 with the increased age (Figure 6a and 6b). The average ratios of in vivo and ex vivo images were shown in Figure 6e and 6f. These results suggested that the concentrations of MAO-B enhanced with age. PD is a common neurodegenerative that loss of dopaminergic neurons near the substantia nigra pars compacta (SNpc) of the midbrain where is a region possessed high concentration of MAO-B. The products of MAO-catalysed reactions may contribute to neurotoxicity and accelerate cell


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Figure 6. Imaging of MAO-B levels in the mice brain with different ages. Fluorescence collection windows: channel 1: 760 - 840 nm, λex = 730 nm. Channel 2: 700 – 800 nm, λex = 650 nm. All the BALB/c mice were incubated MitoCy-NH2 (100 µM, 50 µL in 1:99 DMSO/ saline, v/v) for 30 min with intracranial injection. a) In vivo imaging of mice in group a – d, the age of the mice were 1, 6, 12, and 24-month-old, respectively; b) Ex vivo imaging of separated organs sacrificed from the mice in a); c) Imaging of fresh mice SNpc from b). Two fluorescence collection windows, channel 1: 750 – 800 nm (λex = 730 nm); channel 2: 700 – 740 nm (λex = 650 nm). d) and g) Western blotting analysis for MAO-B levels of mice in a). e) The average fluorescence ratios for a); f) The average fluorescence ratios for b).

treatment of PA or selegiline in mice models. The 15-month old BALB/c mice were divided into 3 groups (Figure 7). Mice in Group a were as control. Mice in group b were treated with PA (100 µM, 8 µL in 1:99 DMSO/ saline, v/v) with intracranial injection for 1.5 h before the injection of our probe. Mice in group c were fed with selegiline hydrochloride tables at 5 mg/kg dosage two times/day for 2 weeks. Then all the mice in the 3 groups were treated with MitoCy-NH2 via intracranial injection. After 30 min, the images were obtained using Bruker In-vivo Imaging System (Figure 7a). The average fluorescence ratios were shown in Figure 7c. We also acquire the images of fresh mice SNpc isolated from mice in a (Figure 7b). The results demonstrated that the treatment of mice by PA or selegiline could obviously decrease the fluorescence intensity in channel 2, revealing the inhibited activity of MAO-B. Our probe was potentially applied for not only the detection of MAO-B activity but also for efficacy evaluation and drug screening.

Conclusion In summary, we develop two NIR fluorescent probe MitoCy-NH2 and MitoHCy-NH2 for detection of the activity of MAO-B and ROS produced from its enzymatic reaction in cell model and mice aging model. The probe MitoHCy-NH2 can provide synergistic detection of MAO-B and ROS produced from its enzymatic reaction in replicative ageing model. The probe MitoCy-NH2 can offer high selectivity and ratiometric NIR fluorescence response for evaluating the level and activity of MAO-B in H2O2-induced cell ageing model and in mice models. The results reveal that the levels of MAO-B increase along with the process of aging.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI:*** More experimental materials and details, synthesis steps, and Compounds characterization (PDF).

AUTHOR INFORMATION Figure 7. Evaluation of therapeutic effects of PA and selegiline in vivo using MitoCy-NH2. The age of theses BALB/c mice was 15-month-old. Before imaging, the mice were treated with MitoCy-NH2 (100 μM, 50 μL in 1:99 DMSO/saline, v/v) for 30 min. a) Group a: control; Group b: intracranial injected PA (100 μM, 8 μL in 1:99 DMSO/ saline, v/v ) for 1.5h then treated as group a ; Group c: fed Selegiline Hydrochloride Tables of 5 mg/kg dosage two times/day for 2 weeks (dissolved the tablet in saline) after 1 hour of gavage treated as group a. Fluorescence collection windows: channel 1: 760-840 nm, λex = 730 nm; Channel 2: 700-800 nm, λex = 650 nm. b) Fluorescence imaging for slices of fresh mice SNpc isolated from a). Fluorescence collection windows: channel 1: 770-810 nm, λex = 730 nm; channel 2: 700 - 740nm, λex = 650 nm. c) The average fluorescence ratios for a).

apoptosis.4 To direct imaging of MAO-B levels, the slice of the fresh mice SNpc in group a – d were made using a vibration slicer (Figure 6c). The fluorescence changes were consistent with Figure 6a and 6b. The expression of MAO-B in SNpc of the four groups were also measured via western blotting assays (Figure 6d and 6g). The results confirmed the trends of MAO-B. And the probe MitoCy-NH2 could be used to image the level changes of MAO-B in vivo. At present, one of the treatment method for PD disease in clinical is the combination of dopamine and selegiline.3 We next utilized the probe MitoCy-NH2 to evaluate activity of MAO-B after the

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

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We thank the National Nature Science Foundation of China (No. 21775162, No. 21405172, No. 41776110, and No. 21575159), the program of Youth Innovation Promotion Association, CAS (Grant 2015170), State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, CAS (Grant KF2016-22), and the Instrument Developing Project of the Chinese Academy of Sciences (YZ201662).

REFERENCES (1) Robert, K. A.; Brunet-Rossinni, A.; Bronikowski, A. M. Aging Cell 2007, 6, 395-404. (2) Colibus, L. D.; Li, M.; Binda, C.; Lustig, A.; Edmondson, D. E.; Mattevi, A. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 12684-12689.



(3) Youdim, M. B. H.; Edmondson, D.; Tipton, K. F.; Nat. Rev. Neurosci. 2006, 7, 295-309. (4) Barnham, K. J.; Masters, C. L.; Bush, A. I. Nat. Rev. Drug Discov. 2004, 3, 205-214. (5) Mattson, M. P.; Nat. reviews Molecular cell biology 2000, 1, 120-130. (6) Mandel, S.; Weinreb, O.; Amit, T.; Youdim, M. B.H. Brain Research Reviews 2005, 48, 379-387. (7) Yamaguchi, K.; Ueki, R.; Nonaka, H.; Sugihara, F.; Matsuda, T. J. Am. Chem. Soc. 2011, 133, 14208-14211. (8) Qian, L.; Li, L.; Yao, S. Q. Acc. Chem. Res. 2016, 49, 626634. (9) Zhou, JJ. P.; Zhong, B.; Silverman, R. B. Anal. Biochem. 1996, 234, 9-12. (10) Yamaguchi, K.; Ueki, R.; Nonaka, H.; Sugihara, F.; Matsuda, T.; Sando, S. J. Am. Chem. Soc. 2011, 133, 14208-11. (11) Zhou, M.; Panchuk-Voloshina, N. Anal. Biochem. 1997, 253, 169-74. (12) Qian, L.; Li, L.; Yao, S. Q. Acc. Chem. Res. 2016, 49, 626-34. (13) Xu, W.; Zeng, Z.; Jiang, J.; Chang, Y. T.; Yuan, L. Angew. Chem., Int. Edit. 2016, 55, 13658-13699. (14) Zhou, J.; Ma, H. Chem. Sci. 2016, 7, 6309-6315. (15) Tang, Y.; Lee, D.; Wang, J.; Li, G.; Yu, J.; Lin, W.; Yoon, J. Chem. Soc. Rev. 2015, 44, 5003-5015. (16) Gao, M.; Yu, F.; Lv, C.; Choo, J.; Chen, L. Chem. Soc. Rev. 2017, 46, 2237-2271. (17) Kim, T. I.; Jin, H.; Bae. J.; Kim, Y. Anal. Chem. 2017, 89, 10565-10569. (18) Nandhikonda, P.; Heagy, M. D. J. Am. Chem. Soc. 2011, 133, 14972-4. (19) Tan, Y.; Zhang, L.; Man, KH.; Peltier, R.; Chen, G.; Zhang, H.; Zhou, L.; Wang, F.; Ho, D.; Yao, S. Q.; Hu, Y.; Sun, H. ACS. Appl .Mater. Interfaces. 2017, 9, 6796-6803. (20) Dai, Z.; Ge, G.; Feng, L.; Ning, J.; Hu, L.; Jin, Q.; Wang, D.; Lv, X.; Dou, T.; Cui, J.; Yang, L. J. Am. Chem. Soc. 2015, 137, 14488-95. (21) Albers, A. E.; Rawlsa, K. A.; Chang, C. J. Chem. Comm. 2007, 4647-4649. (22) Li, L.; Li, K.; Liu, Y.; Xu, H.; Yu, X. Sci. Rep. 2016, 6, 31217. (23) Lu, Y.; Wang, Y.; Dai, B.; Dai, Y.; Wang, Z.; Fu Z.; Zhu, Q. Chinese Chem. Lett. 2008, 19, 947-950. (24) Chen, G.; Yee, D.J.; Gubernator, N.G.; Sames, D. J. Am. Chem. Soc. 2005, 127, 4544-5. (25) Li, X.; Yu, J.; Zhu, Q.; Qian, L.; Li, L.; Zheng, Y.; Yao, S. Q. Analyst 2014, 139, 6092-5. (26) Kim, D.; Sambasivan, S.; Nam, H.; Kim, K. H.; Kim, J.Y.; Joo, T.; Lee, K. H.; Kim K. T.; Ahn, K. H. Chem. Commun. 2012, 48, 6833-6835. (27) Li, X.; Zhang, H.; Xie, Y.; Hu, Y.; Sun H.; Zhu, Q. Org. Biomol. Chem. 2014, 12, 2033-2036. (28) Wu, X.; Li, L.; Shi, W.; Gong, Q.; Li, X.; Ma. H. Anal. Chem. 2016, 88, 1440-6.

Page 8 of 9

(29) Shen, W.; Yu, J.; Ge, J.; Zhang, R.; Cheng, F.; Li, X.; Fan, Y.; Yu, S.; Liu B.; Zhu, Q. ACS. Appl. Mater. Inter. 2016, 8, 927-35. (30) Wu, J.; Lin, T.; Gallagher, J. D.; Kushal, S.; Chung, L. W.; Zhau, H. E.; Olenyuk, B. Z.; Shih, J. C. J. Am. Chem. Soc. 2015, 137, 2366-74. (31) Wu, X.; Shi, W.; Li, X.; Ma, H. Angew. Chem., Int. Edit. 2017, 56, 15319–15323. (32) Li, L.; Zhang, C.; Chen, G.; Zhu, B.; Chai, C.; Xu, Q.; Tan, E.; Zhu, Q.; Lim, K. L.; Yao, S. Q. Nat. Commun. 2014, 5, 3276. (33) Li, L.; Zhang, C.; Ge, J.; Qian, L.; Chai, B.; Zhu, Q.; Lim, K. L.; Yao, S. Q. Angew. Chem., Int. Edit. 2015, 54, 10821-5 (34) Long, S.; Chen, L.; Xiang, Y.; Song, M.; Zheng, Y.; Zhu, Q. Chem. Commun. 2012, 48, 7164-6. (35) Xiang, Y.; He, B.; Li, X.; Zhu, Q. RSC. ADV. 2013, 3, 4876-4879. (36) Han, X.; Song, X.; Yu, F.; Chen, L. Chem. Sci. 2017, 8, 6991-7002. (37) Lee, M. H.; Kim, J. S.; Sessler, J. L. Chem. Soc. Rev. 2015, 44, 4185-91. (38) Zielonka, J.; Joseph, J.; Sikora, A.; Hardy, M.; Ouari, O.; Vasquez-Vivar, J.; Cheng, G.; Lopez, M.; Kalyanaraman, B. Chem. Rev. 2017, 117, 10043-10120. (39) Smith, R. A.; Hartley, R. C.; Cochemé, H. M.; Murphy, M. P. Trends Pharmacol. Sci. 2012, 33, 341-52. (40) Ross, M. F.; Kelso, G. F.; Blaikie, F. H.; James, A. M.; Cochemé, H. M.; Filipovska, A.; Da Ros, T.; Hurd, T. R.; Smith, R. A.; Murphy, M. P. Biochemistry 2005, 70, 22230. (41) Guo, G.; Ma, L.; Jiang, B.; Yi, J.; Tong, T.; Wang, W. J. Cell. Biochem. 2010, 109, 1000-1005. (42) Kurz, D. J.; Decary, S.; Hong, Y.; Erusalimsky, J.D. J. cell. Sci. 2000, 113, 3613-3622. (43) Han, X.; Wang, R.; Song, X.; Yu, F.; Lv, C.; Chen, L. Biomaterials 2017, 156, 134-146. (44) Chen, X.; Tian, X.; Shin, I.; Yoon, J. Chem. Soc. Rev. 2011, 40, 4783−4804. (45) Foreman, J.; Demidchik, V.; Bothwell, J. H.; Mylona, P.; Miedema, H.; Torres, M. A.; Linstead, P.;Costa, S.; Brownlee, C.; Jones, J. D.; Davies, J. M.; Dolan, L. Nature 2003, 422, 442-6. (46) Yu, F.; Gao, M.; Li, M.; Chen, L. Biomaterials 2015, 63, 93. (47) Huang, Y.; Yu, F.; Wang, J.; Chen, L. Anal. Chem. 2016, 88, 4122-9. (48) Li, P.; Zhang, W.; Li, K.; Liu, X.; Xiao, H.; Zhang, W.; Tang, B. Anal. Chem. 2013, 85, 9877-81. (49) Sun, W.; Guo, S.; Hu, C.; Fan, J.; Peng, X. Chem. Rev. 2016, 116, 7768-7817. (50) Han, X.; Song, X.; Yu, F.; Chen, L. Adv. Funct. Mater. 2017. (51) Yuan, L.; Lin, W.; Zheng, K.; He, L.; Huang, W. Chem. Soc. Rev. 2013, 42, 622-661.


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Ratiometric Near-Infrared Fluorescent Probe for Synergistic Detection

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