Light-Up Probes Based on Fluorogens with Aggregation-Induced

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Light-up probes based on fluorogens with aggregationinduced emission characteristics for monoamine oxidase-A activity study in solution and in living cells Wei Shen, Jiajun Yu, Jingyan Ge, Ruoyu Zhang, Feng Cheng, Xuefeng Li, Yong Fan, Shian Yu, Bin Liu, and Qing Zhu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b10528 • Publication Date (Web): 15 Dec 2015 Downloaded from http://pubs.acs.org on December 22, 2015

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ACS Applied Materials & Interfaces

Light-up probes based on fluorogens with aggregation-induced emission characteristics for monoamine oxidase-A activity study in solution and in living cells Wei Shen, ‡a Jiajun Yu, ‡ b Jingyan Ge, b Ruoyu Zhang, c Feng Cheng,b, d Xuefeng Li,b Yong Fan, a Shian Yu, a Bin Liu,*c Qing Zhu*b a

Department of General Surgery, Jinhua Central Hospital, Jinhua, China, 321000,

b

Institute of Bioengineering, Zhejiang University of Technology, Chaowang Road 18, China

310014 c

Department of Chemical and Biomolecular Engineering, National University of Singapore, 4

Science Drive 4, Singapore 117585 d

Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, 52074 Aachen,

Germany

KEYWORDS Aggregation-induced emission, AIEgen, tetraphenylethylene, monoamine oxidase-A, enzymatic assay

ACS Paragon Plus Environment

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ABSTRACT Fluorogens with aggregation-induced emission (AIEgens) have emerged as a powerful and versatile platform for the development of novel biosensors. In this study, a series of water-soluble fluorescent probes based on tetraphenylethylene (TPE) were designed and synthesized for the detection of monoamine oxidases (MAOs) based on specific interactions between the probes and the proteins. Among the six probes developed, t-TPEM displays a significant fluorescence increase upon introduction of MAOs. Of particular significance is that the fluorescence of t-TPEM in the presence of MAO-A is 21-fold higher than other proteins including MAO-B. Lineweaver–Burk plots reveal that t-TPEM acts as an uncompetitive inhibitor of MAO-A with Ki = 17.1 µM, which confirms its good binding affinity towards MAOA. Furthermore, cell imaging experiment reveals that t-TPEM is able to selectively monitor the activity of MAO-A which is localized in mitochondria of MCF-7 cells.

Introduction Monoamine oxidase (MAO), a mitochondrial flavoprotein, is associated with various important biological functions, such as apoptosis, aging and mitochondrial dysfunction.1-3 MAOA and MAO-B, with 70% sequence identity, are known as the two isoforms of MAO.4,5 They have different cellular and tissue distributions, substrate and inhibitor specificities.6 MAO-A, mostly located in catecholaminergic neurons, prefers to have serotonin, norepinephrine and dopamine as substrates.7,8 MAO-B, an enzyme mainly expressed in astrocytes and serotonergic neurons, predominantly metabolizes benzylamine and phenylethylamine.9,10 MAO-A is preferentially inhibited by clorgyline, whereas MAO-B prefers pargyline, rasagiline and selegiline as the inhibitors.11 MAOs are demonstrated to be crucial proteins to maintain the dynamic balance of neurotransmitters in the body, and the deficiency or over-expression of MAOs in humans is associated with neurodegenerative disturbances, developmental disabilities


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and severe intellectual .12-16 To date, both MAO-A and MAO-B have been identified as targets for anti-depressant and anti-neurodegenerative therapies.17,18 The development of noninvasive and real-time sensing strategies for MAOs under physiological conditions is thus highly desirable. So far, fluorescent method is widely used for MAO detection.19,20 For example, the commercially available Amplex® Red reagent could produce strong fluorescence at 585 nm upon treatment with MAOs and their substrates. The approach is based on the detection of H2O2 which could also be released by other redox enzymes. This makes the reagent not suitable for specific detection of MAOs in living cells or in more complex biological systems. Recently, several fluorogenic probes were reported to monitor MAO activities. The hydroxyl groups of the fluorescent molecules were coupled with recognition units such as 1-methyl1,2,3,6-tetrahydropyridine or propylamine to quench the fluorescence. Once the probes were catalyzed by MAOs, the free dyes would be released to generate fluorescence.21-26 These probes displayed outstanding biocompatibility and excellent selectivity towards MAO-B, and were able to detect high expression of MAO-B in the live cells and tissues of Parkinson’s disease models.21 So far, very few fluorescent probes have been reported to monitor MAO-A activity in living cells.27 Moreover, all the reported MAO probes are based on conventional fluorophores, which suffer from aggregation-caused quenching (ACQ) when the fluorophores are at high concentrations or in aggregated state, which is not desirable for biological applications. 28 The concept of aggregation-induced emission (AIE) coined by Tang and co-workers in the last decade describes an opposite phenomenon to the ACQ effect.29-32 The AIE fluorogens (AIEgens), such as tetraphenylethene (TPE) and its derivatives are almost non-emissive when molecularly dissolved, but they produce strong fluorescence upon aggregation. Due to their unique optical features, these fluorogens have emerged as a powerful and versatile platform for


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the development of novel fluorescent probes.33-38 Herein, by taking advantages of the AIE properties, we report the design and synthesis of the first activity-based AIE probe for sensitive detection and imaging of MAO-A in solution and in living cells.

Results and Discussion As shown in Scheme 1A, the TPE based MAO probe is composed of an N-methyl phenylpyridium, a TPE fluorogen and a C-C double bond linker. The N-methyl phenylpyridium and its analogs are unique inhibitors with high binding affinity towards both MAO-A and MAOB.39-41 TPE, as a large substituent at 4-position of phenylpyridium, is expected to make the probe more selective towards MAO-A.39 The positively charged pyridium moiety is hydrophilic, which should render the probe dispersible in aqueous media, especially at low concentrations. As pyridium moiety is electron deficient, the donor-π-acceptor structure with strong charge transfer characteristics often leads to moderate to low fluorescence in polar media. This, together with the intramolecular rotations (RIR) of phenylene rings in aqueous media, should yield low background fluorescence of the probe in the absence of MAO-A.33 Once the probe enters the active site of MAO-A under physiological conditions and forms complex in the hydrophobic pocket of MAO-A, the reduced environmental hydrophilicity and restricted intramolecular rotations should turn on the probe fluorescence. The schematic illustration for the fluorescence turn-on detection of MAO-A is shown in Scheme 1B.


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Scheme 1. a) The structure of t-TPEM and b) the proposed sensing mechanism of t-TPEM to MAO-A. To gain insight into the binding affinity and fluorescence increment of the probe towards MAOs, methyl group, 2-ethylhydroxyl group and benzyl group, which have different solubility and volume, were chosen as the R substituent groups. The six probes were synthesized as shown in Scheme 2. p-Tolylboronic acid and 4-bromopyridine were reacted to form 4-(p-tolyl)pyridine (1) in 50% yield using palladium catalyzed coupling reaction. Bromination of 1 in the presence of NBS/BPO led to compound 2 in 60% yield,42 which was then converted to the corresponding aldehyde 3 in the presence of hexamethylenetetramine (HMTA). Subsequent condensation reaction between 3 and TPE gave the mixture of cis/trans isomers of 4, which were further treated with methyl iodide (MeI for TPEM) or BrCH2CH2OH (for TPEA), or BrCH2C6H5 (for TPEB ) and followed by flash column purification to afford the isolated cis- and trans- probes as c-TPEM, c-TPEA, c-TPEB, t-TPEM, t-TPEA, and t-TPEB, respectively. Experimental details and characterizations are shown in the Supporting Information.


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Scheme 2. Synthesis of TPE based monoamine oxidase probes. Reagents and Conditions: a: Pd(OAc)2, PPh3, NaHCO3, DMF, rt.; b: NBS/BPO; c: (i) HMTA in DMF; (ii) AcOH:H2O = 1:1; d: t-BuONa, TPE; e: CH3I, r.t. or BrCH2CH2OH, or BrCH2C6H5. The excitation and emission spectra of these six probes were investigated in THF and Tris buffer (50 mM, pH 7.4), respectively and the results are shown in Figures 1a and S1. c-TPEM and t-TPEM (10 µM) display excitation maxima at 340 and 360 nm and exhibit fluorescence emission maxima at 540 and 560 nm, giving Stokes shifts of 200 nm. In comparison, c-/t-TPEA have slightly blue-shifted excitation maxima at 330/360 nm and emission maxima at 500/525 nm. The excitation and emission maxima of c-/t-TPEB were observed at 385/395 nm and 565/605 nm, respectively. Interestingly, among the six probes, trans- forms experience bathochromic effect compared with their cis- counterparts. To test whether the probes remain AIE active, the emission spectra of c-TPEM and t-TPEM in THF-acetonitrile (ACN) at different ratios were studied. With the increased THF content, both probes show gradually red-


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shifted emission maxima. We also noticed that, with the addition of THF to ACN, the fluorescent signal increased slightly when the THF content was from 0-90%, but the increase became dramatic when up to 90% THF was added to the ACN solution (Figure S2). This property is in accordance with reported AIE dyes, suggesting that c-TPEM and t-TPEM have both intramolecular charge transfer and AIE characteristics. 43 The AIE properties of c-/t-TPEAs and c-/t-TPEBs were confirmed in the same way and the results are shown in Figure S2. To test the probe response to MAOs, all six probes (10 µM) were treated with MAO-A and MAO-B, respectively. To our surprise, only methyl substituted probes: c-TPEM and t-TPEM display significant fluorescence increase, while the other four probes c-TPEA, c-TPEB, tTPEA, and t-TPEB only show slight response toward both MAO-A and MAO-B, indicating that the interactions are negligible (Figure 1b). This is because the large substituent group at the Nposition will reduce the binding affinity between the probe and the protein, which is in consistent with our previous results.26 Therefore, c-TPEM and t-TPEM were chosen for further studies.

a

b

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t-TPEM c-TPEM t-TPEA c-TPEA t-TPEB c-TPEB

600

Relative Intensity

Relative Intensity

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400 500 600 Wavelength(nm)

700

450 300 150 0

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MAO B

Figure 1. a) The normalized excitation spectra of t-TPEM and c-TPEM (10 µM) in THF and their emission spectra in Tris buffer (50 mM, pH 7.4). (olive: c-TPEM excitation, red: t-TPEM excitation; blue: c-TPEM emission, black: t-TPEM emission); b) The fluorescence intensity of


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six probes (10 µM) upon interaction with MAO-A and MAO-B; Excitation at 360 nm, emission at 540 nm. To verify their inhibitory activities towards MAOs, both the half maximal inhibitory concentration (IC50) and inhibition constant (Ki) values were determined using our reported fluorogenic in vitro assays.44 The IC50 values were calculated by plotting percent activity versus log[concentration of probes]. Both probes show good binding activity toward MAO-A with IC50 values in the micromolar range, while the IC50 values for MAO-B are in millimolar to molar range, which give an excellent selectivity of over 1000 between MAO-A and MAO-B (Figure S3, Table 1). In addition, the Ki values further confirm that, in comparison with c-TPEM, tTPEM displays higher selectivity to MAO-A. To obtain the kinetic parameters for c-TPEM and t-TPEM, the michaelis constants (Km) and the maximum initial velocities Vmax for MAO-A and B were determined from the Lineweaver–Burk plots, which are summarized in Table S1. Although Vmax values of both probes are less than those of the known MAO probe CouMAO (Vmax values for MAO-A and MAO-B are 1.3 and 5.4 µM/mg/s, respectively)19, the Km values of t-TPEM (58.8 and 86.6 µM for MAO-A and MAO-B respectively, Table S1 ) are similar to that of CouMAO (62.4 and 83.5 µM for MAO-A and MAO-B, respectively), indicating that tTPEM has enzymatic affinity as good as that of CouMAO. Lineweaver–Burk plots (Figure 2) also reveal that t-TPEM could act as an uncompetitive inhibitor of MAO-A with Ki = 17.1 µM. These results confirm that t-TPEM has good binding affinity towards MAO-A, which suggests an activity based mechanism for MAO-A detection.


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50 t-TPEM 1/Rate (RFU/s)

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40 30 20 10 0 0.00

0.03 0.06 0.09 1/Substrate (µM)

0.12

Figure 2. Lineweaver–Burk plots of MAO-A for different concentrations of t-TPEM (0-50 µM). Table 1 The monoamine oxidase inhibitory activity of the probes IC50

Ki

Probe MAO-A

MAO-B

MAO-A

MAO-B

c-TPEM

77.0 μM

9.0 mM

39.9 μM

50.3 μM

t-TPEM

51.9 μM

7.1 M

17.1 μM

101.8 μM

Next, MAO assays with c-TPEM and t-TPEM were carried out in Tris buffer (pH 7.4, 50 mM), and the fluorescence spectra were collected with microplate reader at excitation/emission wavelength (λex/λem ) = 360/540 nm, 360/560 nm for c-TPEM and t-TPEM, respectively (Figure 3a). For both probes, little fluorescence enhancement was observed in the presence of MAO-B, whereas fluorescent signals increased immediately upon addition of MAO-A. The results are essentially in agreement with their inhibitory activities towards MAO-A and MAO-B. To further verify the interactions between MAO-A and the probes, the fluorescence intensities of t-TPEM (10 µM) upon incubation with different concentrations (0 to 117 µg) of MAO-A were also monitored. As shown in Figure 3b, the signals increase gradually with the addition of MAO-A,


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demonstrating that the fluorescence of t-TPEM is turned on. Meanwhile, we also notice that the emission intensity of t-TPEM incubated with MAO-A is significantly higher than that of cTPEM (10 µM). The 40-fold enhancement of the probe signal at the emission maximum (560 nm) for t-TPEM verus 20-fold at 540 nm for c-TPEM indicates that t-TPEM possesses better selectivity to MAO-A than c-TPEM does (Figure S3). The increase in emission intensity of tTPEM is due to its specific binding with the hydrophobic pocket of MAO-A containing Tyr-69, Asn-181, Phe-208, Val-210, Gln-215, Cys-323, Ile-325, Ile-335, Leu-337, Phe-352, Tyr-407, and Tyr-444. 45 Subsequently, time-dependent enzymatic reactions between MAO-A (or MAO-B) and t-TPEM were studied by monitoring the emission intensity changes at 560 nm. The fluorescence intensities increase with extended time and saturate in 5 min (Figure 3c). The limit of detection (LOD) values for MAO-A are 17.8 and 7.1 µg mL-1 using c-TPEM and t-TPEM, respectively, which are comparable with the commercial assay kit.21 To confirm the specific interaction between c-TPEM/t-TPEM and MAO-A, clorgyline (10 µM), a well-known MAO-A inhibitor was incubated with the protein for 1 h before probe treatment. No obvious fluorescence enhancement is observed in the presence of the inhibitor (Figure 3d), which further proves the specific interaction between both probes and MAO-A. To further examine the selectivity of t-TPEM towards MAO-A, the probe (10 µM) was treated with a panel of different proteins, including MAO-B, BSA, papain, proteinase K, pepsin, chymotrypsin, and trypsin alkali phosphatase in 50 mM Tris buffer. A low fluorescence enhancement was found for the non-specific proteins. t-TPEM displays a 21-fold higher fluorescent signal than other proteins, indicating the high selectivity of the probe (Figure 3d). When similar experiments were conducted for c-TPEM, a 4-fold brighter signal is observed for MAO-A relative to other proteins. The results indicate that t-TPEM has higher sensitivity


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towards MAO-A than c-TPEM, although both are able to differentiate MAO-B from MAO-A. Therefore, t-TPEM was further used for the following cell imaging study.

500 400 300 200 100 0 450

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0 µg

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papain proteinase K pepsin chymotrypsin trypsin

ALP BSA MAO-B MAO-A

c-TPEM

t-TPEM

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t-TPE+MAO-A t-TPE+MAO-B t-TPE c-TPE+MAO-A c-TPE+MAO-B c-TPE

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Fluorescence Intensity


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0

50 100 150 200 250 300 Time (sec)

0.0

Figure 3. a) The emission spectra of c-TPEM and t-TPEM (10 µM) upon interaction with 80 µg/mL MAO-A and MAO-B; b) The emission spectra of 10 µM t-TPEM after treatment with different amount of MAO-A (0 to 117 µg). c) The time course of fluorescence intensity of cTPEM and t-TPEM (10 µM) upon treatment with 80 µg/mL MAO-A and MAO-B; d) The (II0)/I0 values of c- and t-TPEM upon addition of a panel of 80 µg/mL proteins (the fluorescence intensity after protein addition: I, the initial fluorescence intensity before protein addition: I0) in Tris buffer (pH 7.4, 50 mM) at 37 oC. Excitation at 360 nm, emission at 540 nm for c-TPEM or 560 nm for t-TPEM.

Subsequently, docking experiments were carried out to gain better insights into the recognition of t-TPEM towards human MAO-A and MAO-B. t-TPEM was docked into the ligand binding


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pocket of both MAO-A (PDB: 2Z5X) and MAO-B (PDB:1S3E) by YASARA molecular modeling program under AMBER03 force field. The docking result was shown in Figure 4. The methyl pyridinium of t-TPEM is ideally accommodated in the ‘aromatic cage’ composed by covalent FAD coenzyme and two tyrosine (Tyr407 and Tyr444) residues. Especially, pyridinium forms a T-shape orientation with FAD and builds a parallel-displaced stacking interaction47 with two tyrosine residues, and the interactions between the cage and pyridinium ring are the major contributors to the formation of MAO-A and t-TPEM complex. The binding energy between tTPEM and MAO-A is -7.06 kcal/mol. Four benzene rings of t-TPEM lay in the substrate/inhibitor entrance of MAO-A surrounded by three different loops (red color, residues Val93-Glu95, Tyr109-Pro112, and Phe208-Asn212). Importantly, these loops, causing a shorter and wider substrate cavity of MAO-A than that of MAO-B, result the significant structural differences between MAO-A and MAO-B.48 t-TPEM is difficult to enter the substrate cavity of MAO-B, thus, no positive docking results were obtained between t-TPEM and MAO-B (the binding energy between t-TPEM and MAO-B is above zero). These docking studies again provide stronger evidence that t-TPEM has higher inhibition towards MAO-A than MAO-B.


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Figure 4. Binding mode of t-TPEM in the ligand binding pocket of MAO-A. MAO-A is represented in cartoon format. t-TPEM, FAD, Tyr407 and Tyr444 are shown in sticks. Three loops in substrate/inhibitor entrance of MAO-A are highlighted in red color.

We next used fluorescence confocal microscopy to investigate the potential of t-TPEM for imaging of MAOs activity in live cells. Breast cancer MCF-7 cells were selected due to their high expression level of MAO-A.46 As MAOs are located in the mitochondria, it is thus of high importance to check whether the probe could stain mitochondria. t-TPEM was further evaluated for its ability to specifically localize in mitochondria using MitoTracker®Red as the co-stain. Upon incubation 1 µM of t-TPEM with MCF-7 in the presence or absence of MAO-A inhibitor (200 µM clorgyline), the probe was excited at 405 nm and the emission was monitored at 550650 nm. The confocal imaging results are shown in Figure 5. No background fluorescence is observed for cells in the absence of the probe (Figure 4a). Upon incubating the cells with tTPEM solution (1 µM) for 1 h, the fluorescence images were collected upon excitation at 405 nm and the emission signal was collected at 560 ± 30 nm. As shown in Figure 4d, strong fluorescence from the cells is observed. However, when the cells were pretreated with 200 µM of clorgyline (MAO-A inhibitor,) the cell fluorescence from t-TPEM is almost quenched, which indicates specific interaction between the probe and MAO-A. To examine cellular localization, MitoTracker®Red (50 nM) was incubated with t-TPEM and the overlay image (Figures 4e and 4f) show an excellent overlap. This indicates that t-TPEM is able to efficiently and selectively detect MAO-A which localizes in the mitochondria of living cells. These results provide direct evidence that t-TPEM is a suitable probe for imaging of MAO activity in live cells.


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MitoTracker®Red

t-TPEM

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Merged

a

b

c

d

e

f

g

h

i

MCF-7 cells + MT

-inhibitor

+ inhibitor

Figure 5. Confocal fluorescence images of MCF-7 cells upon treatment with t-TPEM (1 µM, 2h) in the absence (d-f) or presence (g-i) of MAOs inhibitor clorgyline (200 µM, 1h). Green = tTPEM; Red = MitoTracker®Red; Blue = Nuclei stain. (a-c): cells only treated with DMSO and MitoTracker®Red (50 nM). All images share the same scale bar: 10 µm. Insets are the overlay of bright field images with nuclear stain. Excitation and emission wavelengths: 405 nm and 550650 nm for t-TPEM; 580 nm and 590-605 nm for MitoTracker®Red; 405 nm and 450-475 nm for Hoechst nuclei stain. Conclusion In summary, six cis- and trans- probes c-TPEM, c-TPEA, c-TPEB, t-TPEM, t-TPEA, and tTPEB based on tetraphenylethylene (TPE) with aggregation-induced emission were designed and synthesized for the detection of monoamine oxidases (MAOs). Among the six probe developed, methyl substituted probe TPEMs displays a significant fluorescence increase upon introduction of MAOs. Particularly, the fluorescence of trans isomer: t-TPEM display 21-fold higher than other proteins including MAO-B. Lineweaver–Burk plots revealed that t-TPEM


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acted as an uncompetitive inhibitor of MAO-A with Ki = 17.1 µM, which confirmed that it has good binding affinity towards MAO-A. Furthermore, cell imaging experiments revealed that tTPEM was able to efficiently and selectively detect the activity of MAO-A which was localized in mitochondria of cells, which offers a new opportunity for convenient high-throughput MAOsrelated drug screening in the near future. Experimental Section Materials and Instruments Compounds were visualized by UV light (254 and 365 nm) and all reactions were monitored by thin layer chromatography (TLC). Column chromatography was performed on silica gel (200-300 mesh). 1H NMR (400 MHz or 300 MHz) and

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C NMR (101

MHz or 75 MHz) spectra were recorded on a Bruker instrument Data for 1H NMR are reported as follows: chemical shift (δ, ppm), multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet, q = quartet or unresolved, coupling constant (J) in Hz, integration). Data for 13C NMR were recorded in terms of chemical shift (δ, ppm) relative to the center of the triplet at 77.0 ppm for CDCl3. Mass spectra (MS) were measured with Bruker instrument. Fluorescence spectra were determined on a Multi-Mode Microplate Readers. Mass spectra were obtained on Bruker ESI-MS system. Imaging was done with the Leica TCS SP5X confocal microscope system equipped with Leica HCX PL APO 63×/1.20 W CORR CS, 405 nm diode laser, white laser (510−600 nm, with 1 nm increments, with eight channels AOTF for simultaneous control of eight laser lines.), and a photomultipliertube (PMT) detector ranging from 510 to 600 nm for steady state fluorescence measurement. Images were processed with Leica Application Suite Advanced Fluorescence (LAS AF). Human recombinant Human recombinant Monoamine Oxidase A (M7316) and B (M7441) (5 mg/mL), chymotrypsin (C7762), trypsin (T8003), Alkaline phosphatase (P5931), clorgyline (M3778) was purchased from Sigma Aldrich. Papain


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(416760100), proteinase K (E080121), pepsin (P0103), Bovine Serum Albumin (BSA, 134730100) were purchased from Energy Chemical. Synthesis Synthesis of 4-(p-tolyl)pyridine (1). 4-Bromo-pyridine hydrochloride (0.72g, 3.7mmol) and p-tolylboronic acid (0.55g, 4.0mmol) were dissolved in 15 mL of DMF, followed by the addition of sodium hydrocarbonate (2.4g, 13.0mmol), Pd(OAc) 2 (83 mg，0.37 mmol), PPh3 (193 mg，4 mmol). The resulting solution was stirred for 18 h at 90-95°C. The reaction mixture was extracted with diethyl ether (100 mL × 3), and the organic layer was washed with water (100 mL × 2), dried over MgSO4 and concentrated in vacuo to afford 1 as a pale yellow oil (31 mg, 50 %). 1

H NMR (400 MHz, CDCl3) δ 8.65 (d, J = 6.1 Hz, 2H), 7.55 (d, J = 8.1 Hz, 2H), 7.50 (dd, J =

4.6, 1.5 Hz, 2H), 7.30 (d, J = 7.9 Hz, 2H), 2.43 (s, 3H); ESI-MS m/z 170.2 (M+1)+. Synthesis of 4-(4-(bromomethyl)phenyl)pyridine (2). N-Bromosuccinimide (17 mg, 0.096 mmol) was added to a solution of 4-(p-tolyl)pyridine (19 mg, 0.11 mmol), carbon tetrachloride (0.5 mL) and benzoyl peroxide (1 mg) and the mixture was heated in a sealed vial at 80 °C for 4 h. Ethyl acetate (10 mL) was added and the organic layer was washed with water (2 mL × 2), and dried over MgSO4. The solvent was removed under reduced pressure at 0 °C to afford crude 2 (16 mg, 60%). 1H NMR (CDCl3, 400 MHz) δ 8.73 (s, 1H), 8.09 (d, J = 7.6 Hz, 2H), 7.73 – 7.60 (m, 3H), 7.59 – 7.51 (m, 2H), 7.43 (t, J = 7.5 Hz, 2H); ESI-MS m/z 248.2 (M+1)+, 250.2 (M+1)+ Synthesis of 4-(pyridin-4-yl)benzaldehyde (3) 4-(4-(Bromomethyl)phenyl)pyridine (54 mg, 0.12 mmol) was dissolved in CHCl3 (5 mL), and hexamethylenetetramine(methenamine) (HMTA, 51.7 mg, 0.37 mmol) was added slowly at 0 ºC. The mixture was allowed to warm up to room temperature and reflux for 2 h. The mixture of AcOH/H2O = 1:1 (6 mL) was added and the


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mixture was further refluxed for 2 h. The reaction was neutralized with saturated sodium carbonate solution (100 mL). The aqueous phase was extracted with ethyl acetate (EtOAc, 300 mL×2), and the organic layer was dried over MgSO4. The solvent was removed by rotary evaporation and to yield 3 as a white solid (12 mg, 82% yield). 1H NMR (CDCl3, 400 MHz) δ 10.11 (s, 1H), 8.74 (dd, J = 4.5, 1.6 Hz, 2H), 8.04 – 8.01 (m, 2H), 7.82 (d, J = 8.2 Hz, 2H), 7.56 (dd, J = 4.5, 1.7 Hz, 2H). ESI-MS m/z 184.2 (M+1)+ Synthesis of compound 4. 4-(Pyridin-4-yl)benzaldehyde (156 mg, 0.85 mmol) and tetraphenylethene (600 mg, 0.85 mmol) are dissolved in DMF (5 mL), and t-BuOK (48 mg， 0.428 mmol) was added slowly at 0 ºC. The mixture was stirred overnight at room temperature. After complete consumption of the starting material monitored using TLC plate, the resulting solution was diluted with ice-water (20 mL). The aqueous phase was extracted with EtOAc (300 mL × 2), and the organic layer was dried over MgSO4. The solvent was removed by rotary evaporation and the crude product was purified by flash chromatography using petroleum ether/ethyl acetate (3:1) as the eluent to yield 4 as a pale-yellow solid. 1H NMR (CDCl3, 400 MHz) cis-isomer (white solid, 81 mg, 27% yield): δ 8.67 (s, 2H), 7.51 (ddd, J = 12.3, 5.8, 4.6 Hz, 4H), 7.34 (dd, J = 13.8, 8.2 Hz, 2H), 7.18 – 6.99 (m, 12H), 6.97 – 6.87 (m, 6H), 6.58 (qd, J = 12.2, 7.3 Hz, 2H), 2.26 (s, 3H); trans-isomer (white solid, 109 mg, 36.3% yield): 1H NMR (400 MHz, CDCl3) δ 8.67 (d, J = 4.4 Hz, 2H), 7.65 (dd, J = 8.3, 2.2 Hz, 2H), 7.60 (dd, J = 8.3, 4.6 Hz, 2H), 7.54 (d, J = 4.7 Hz, 2H), 7.33 – 7.29 (m, 2H), 7.16 – 7.02 (m, 14H), 6.97 – 6.90 (m, 4H), 2.28 (t, J = 6.2 Hz, 3H); ESI-MS m/z 526.4 (M+1)+ . Synthesis of c-/t-TPEM. Methyl iodide (MeI, 23 µL, 0.37 mmol) was added to the cis- or trans- isomers of 4 (53 mg, 0.1 mmol) and K2CO3 (57 mg, 0.4 mmol) in 5 mL of DMF at room temperature. After refluxing for 6 hours, the resulting solution was concentrated under reduced


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pressure and the crude product was purified by flash chromatography using petroleum ether/ethyl acetate (1:3) to yield c-TPEM as a pale-yellow solid (59 mg, 89%). 1H NMR (400 MHz, CDCl3) δ 9.21 (dd, J = 6.9, 2.0 Hz, 2H), 8.17 (d, J = 6.7 Hz, 2H), 7.63 (dd, J = 8.2, 5.0 Hz, 2H), 7.43 (dd, J = 17.6, 8.4 Hz, 2H), 7.16 – 6.99 (m, 12H), 6.96 – 6.89 (m, 6H), 6.68 (dd, J = 12.2, 6.3 Hz, 1H), 6.53 (dd, J = 12.2, 6.0 Hz, 1H), 4.67 (s, 3H), 2.27 (d, J = 12.4 Hz, 3H);

13

C NMR (101 MHz,

CDCl3) δ153.56, 145.60, 145.60, 143.34, 143.05, 142.90, 140.95, 140.75, 140.16, 139.72, 135.87, 134.62, 132.04, 132.04, 131.88, 130.75,130.75, 130.75, 130.75, 130.75, 130.75, 130.75, 130.75, 129.77, 129.77, 128.91, 128.50, 128.50, 127.89, 127.89, 127.89, 127.89, 127.89, 127.89, 127.89, 126.63, 126.63, 123.75, 123.75, 47.12, 20.78. HR-MS (ESI): m/z 540.2702 [(M-I)+, calcd. 540.2686]. t-TPEM Yellow solid, 54 mg (81%).1H NMR (400 MHz, CDCl3) δ 9.15 (d, J = 6.0 Hz, 2H), 8.18 (dd, J = 7.0, 2.2 Hz, 2H), 7.74 (dd, J = 8.5, 2.6 Hz, 2H), 7.55 (dd, J = 8.5, 3.4 Hz, 2H), 7.26 – 7.20 (m, 2H), 7.14 – 6.95 (m, 14H), 6.93 – 6.87 (m, 4H), 4.58 (s, 3H), 2.24 (d, J = 5.2 Hz, 3H);

13

C NMR (101 MHz, d6-DMSO) δ153.55, 145.49, 145.49, 143.43, 143.43,

143.21, 141.03, 140.88, 140.26, 139.77, 135.83, 134.75, 132.00, 131.20, 131.20, 130.91, 130.91, 130.69, 130.69, 130.69, 130.69, 130.69, 130.69, 128.52, 128.52, 128.52, 127.92, 127.92, 127.92, 127.79, 127.50, 127.50, 127.09, 126.66, 126.41, 126.41, 123.55, 123.55, 47.00, 20.74. HR-MS (ESI): m/z 540.2637 [(M-I)+, calcd. 540.2686]. Synthesis of c-/t-TPEB. Compound 4 (27 mg, 0.05 mmol) and KI (16.6 mg, 0.1 mmol) was placed in a flask which was flushed with nitrogen, and degassed acetone (5 mL) was added. The solution was cooled to 0℃, and benzylbromide (10.4 mg, 0.06mmol) was added by a syringe. The solution was stirred at 0℃ for 4h, and the stirring was continued at room temperature overnight. The solvent was evaporated in vacuo and the crude product was purified by flash chromatography using petroleum ether/ethyl acetate (3:1) as the eluent to afford c or t-TPEB. c-


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TPEB: pale-yellow solid, 30 mg (78%). 1H NMR (400 MHz, CDCl3) δ 9.55 (s, 2H), 8.11 (d, J = 6.4 Hz, 2H), 7.70 (d, J = 5.3 Hz, 2H), 7.59 (d, J = 8.4 Hz, 2H), 7.38 (d, J = 11.6 Hz, 5H), 7.21 – 6.89 (m, 18H), 6.66 (s, 1H), 6.53 (s, 1H), 6.23 (s, 2H), 2.26 (s, 3H).

13

C NMR (101 MHz,

CDCl3) δ155.67 (s), 144.81 (s), 143.88 (s), 143.55 (d, J = 8.0 Hz), 142.03 (s), 141.27 (s), 140.50 (d, J = 17.2 Hz), 139.84 (s), 136.04 (s), 134.19 (s), 133.10 (d, J = 19.2 Hz), 131.76 – 130.80 (m), 130.28 (s), 129.58 (t, J = 10.6 Hz), 128.50 – 127.70 (m), 127.61 (d, J = 7.2 Hz), 126.34 (s), 124.30 (s), 63.31 (s), 21.16 (d, J = 10.4 Hz). HR-MS (ESI): m/z 616.2984 [(M-I)+, calcd. 616.2999]. t-TPEB: pale-Yellow solid, 29 mg (75.4%).1H NMR (400 MHz, CDCl3) δ 9.36 (d, J = 6.6 Hz, 2H), 8.16 (dd, J = 6.9, 2.3 Hz, 2H), 7.72 (dd, J = 8.5, 2.9 Hz, 2H), 7.69 – 7.64 (m, 2H), 7.54 (dd, J = 8.5, 4.3 Hz, 2H), 7.40 – 7.30 (m, 4H), 7.25 (dd, J = 11.4, 8.4 Hz, 2H), 7.12 (dd, J = 4.8, 2.0 Hz, 2H), 7.11 – 7.09 (m, 4H), 7.07 (dd, J = 4.7, 3.2 Hz, 2H), 7.05 (s, 1H), 7.04 – 7.02 (m, 3H), 6.99 – 6.95 (m, 1H), 6.93 (d, J = 6.4 Hz, 2H), 6.91 (s, 2H), 6.11 (s, 2H), 2.27 – 2.22 (m, 3H).

13

C NMR (101 MHz, CDCl3) δ 155.35 (s), 144.46 (s), 143.68 (dd, J = 16.6, 6.7 Hz),

141.45 (s), 140.54 (d, J = 7.8 Hz), 139.82 (s), 136.23 (d, J = 13.5 Hz), 134.23 (s), 132.79 (s), 131.80 (d, J = 5.0 Hz), 131.51 – 130.98 (m), 130.29 (s), 129.88 (s), 129.54 (d, J = 6.8 Hz), 128.72 – 128.09 (m), 127.63 (dd, J = 14.1, 5.4 Hz), 126.59 – 126.01 (m), 124.13 (s), 63.15 (s), 21.12 (s).

Synthesis of c-/t-TPEA. Compound 4 (27 mg, 0.05 mmol) and KI (16.6 mg, 0.1 mmol) were dissolved in toluene, and 2-bromothanol (9.4 mg, 0.075 mmol) was added. The mixture was refluxed for 12 hours. After reaction, the solvent was evaporated in vacuo and the crude product was purified by flash chromatography using petroleum ether/ethyl acetate (3:1) as the eluent to afford t-TPEA as a white solid (15 mg, 43%). 1H NMR (400 MHz, CDCl3) δ 9.00 (s, 2H), 8.15 (s, 2H), 7.75 (s, 2H), 7.50 (s, 2H), 7.22 (s, 2H), 7.17 – 6.99 (m, 13H), 6.94 (d, J = 4.1 Hz, 5H),


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4.91 (s, 2H), 4.22 (s, 2H), 2.27 (d, J = 16.0 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ155.33 (s), 144.73 (s), 144.46 (s), 143.58 (s), 141.54 (d, J = 18.8 Hz), 140.55 (s), 139.83 (s), 136.28 (s), 134.18 (s), 131.40 (t, J = 22.4 Hz), 131.12 – 129.90 (m), 128.04 (dd, J = 58.0, 9.3 Hz), 126.26 (d, J = 14.9 Hz), 124.09 (s), 62.71 (s), 60.65 (s), 21.15 (s). HR-MS (ESI): m/z 570.2712 [(M-I)+, calcd. 570.2791]. c-TPEA: white solid, 14 mg (40%).1H NMR (400 MHz, CDCl3) δ 9.00 (d, J = 20.8 Hz, 2H), 8.02 (dd, J = 17.3, 6.4 Hz, 2H), 7.65 (d, J = 8.5 Hz, 1H), 7.47 (d, J = 8.4 Hz, 2H), 7.00 (d, J = 2.6 Hz, 5H), 6.98 – 6.92 (m, 6H), 6.91 – 6.85 (m, 3H), 6.85 – 6.79 (m, 5H), 6.53 (d, J = 16.4 Hz, 1H), 6.39 (s, 1H), 4.87 (d, J = 19.2 Hz, 2H), 4.09 (s, 2H), 2.17 (t, J = 5.9 Hz, 3H). 13

C NMR (101 MHz, CDCl3) δ 155.44 (s), 154.74 (s), 153.45 (s), 152.35 (s), 150.48 (s), 146.91

(s), 146.55 (s), 145.39 (d, J = 146.3 Hz), 143.53 (s), 141.68 (s), 141.34 (s), 139.71 (s), 138.12 (s), 136.07 (s), 133.50 (d, J = 119.1 Hz), 132.70 – 132.37 (m), 132.70 – 132.37 (m), 131.48 (d, J = 50.0 Hz), 130.25 (s), 128.64 (s), 128.32 (d, J = 15.5 Hz), 127.80 (d, J = 50.3 Hz), 126.54 – 126.42 (m), 125.15 (d, J = 236.6 Hz), 62.67 (s), 60.64 (s), 21.32 (s).

Titrations of probes with MAOs The stock solution of each probe was prepared in DMSO (10 mM). They were diluted with Tris buffer (50 mM, pH 7.4) to make 10 µM working solution, and then MAO-A or MAO–B (80 µg/mL) was added and the mixture was incubated for 1 hour at 37 °C. The emission spectra were recorded from 450 to 750 nm upon excitation of 360 nm. The selectivity of c-TPEM and t-TPEM was studied by incubation with other proteins including MAO-B, BSA, papain, proteinase K, pepsin, chymotrypsin, and trypsin alkali phosphatase in 50 mM Tris buffer. Inhibitory activity of probes towards MAOs The IC50 values of MAO-A or MAO-B were assessed by a series concentrations of c-TPEM or t-TPEM (0-200 µM) incubated with MAO-A


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or MAO-B (80 µg/mL) at 37 °C in Tris buffer (50 mM, pH = 7.4). Then the activities of MAOs were assayed by the reported method.44 Kinetic study of probes In 96-well black flat-bottom plates, c-TPEM or t-TPEM (0 µM to 50 µM) and MAO-A or MAO-B (final concentration of 80 µg/mL) were added to 300 µL borate buffer (50 mM, pH 8.4). Nine different concentrations of CouMAO (10, 12, 17, 25, 50, 80, 100, 150, 200 µmol/L) were introduced to each plate. The assay was monitored at 37°C, and the fluorescence spectra were collected at λex/λem of 360/460 nm every 3 min. Live Cell imaging MCF-7 cells were seeded in glass bottom dishes (Mattek) and incubated for 24 h. On the second day, the cells were treated with t-TPEM (1 μM, 0.1% DMSO in growth medium). After 2 h, the growth medium was removed, and cells were gently washed twice with medium. The inhibition experiment was carried out under pretreatment with MAO inhibitor clorgyline (200 µM, 0.1% DMSO in growth medium) for 2 h before the addition of t-TPEM. Subsequently, cells were incubated with 250 nM of MitoTracker®Red and 100 nM Hoechst 33342 nucleic acid stain. Finally, cells were then washed twice with medium and fluorescence imaging experiments were performed on a Leica TCS SP5X Confocal Microscope. Protein (receptor) and ligand preparation for docking All the in silico work was performed on YASARA molecular modeling program (version 15.8.30).49,50 The X-ray crystallographic structures of MAO-A (PDB:2Z5X, resolution of 2.2Å)51 and MAO-B (PDB: 1S3E, resolution of 1.6Å)52 were obtained from the Protein Data Bank. Water molecules, ligands and other hetero atoms were removed from the protein molecule. Missing hydrogen atoms of the proteins were added by the command ‘clean’. Energy minimization of protein was performed to remove bumps and correct the covalent geometry. The ligand molecule (t-TPEM) structure was directly drawn


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in YASARA and followed by an energy minimization. The minimized protein and ligand were saved in SCE and YOB format, respectively, for further docking analysis. Docking analysis Global docking was performed using AutoDock53 under the default docking parameters, and point charges initially assigned according to the AMBER03 force field,54 and then damped to mimic the less polar Gasteiger charges. Subsequently, local docking was executed to predict the binding energy and fine-tune the ligand placement in the binding site.

ASSOCIATED CONTENT Fluorescence spectrum of six probes, concentration dependent inhibition ratio of MAOs by cTPEM and t-TPEM, kinetic parameters of MAOs inhibition by c-TPEM and t-TPEM. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] *E-mail: [email protected] Author Contributions ‡ Authors W. S. and J. J. Y. contributed equally to this work.

Notes The authors declare no competing financial interest.


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ACKNOWLEDGMENT The authors are grateful to the National Nature Science Foundation of China (No. 21272212, 21472172), Project of Science Technology Department of Zhejiang Province (2014C33141), Project of Science Technology Department of Jinhua City (2013–3–003) and the Singapore National Research Foundation Investigatorship (R279-000-444-281) for financial support. REFERENCES 1. Nicotra, A.; Pierucci, F.; Parvez, H.; Senatori, O. Monoamine Oxidase Expression During Development and Aging. Neurotoxicology 2004, 25(1), 155-165. 2. Damier, P.; Kastner, A.; Agid, Y.; Hirsch, E. C. Does Monoamine Oxidase Type B Play a Role in Dopaminergic Nerve Cell Death in Parkinson's Disease? Neurology 1996, 46(5), 12621262. 3. Fowler, J. S.; MacGregor, R. R.; Wolf, A. P.; Arnett, C. D.; Dewey, S. L.; Schlyer, D.; Sachs, H. Mapping Human Brain Monoamine Oxidase A and B with 11C-labeled Suicide Inactivators and PET. Science 1987, 235(4787), 481-485. 4. Lan, N. C.; Heinzmann, C.; Gal, A., Klisak, I.; Orth, U.; Lai, E.; Shih, J. C. Human Monoamine Oxidase A and B Genes Map to Xp11. 23 and are Deleted in a Patient with Norrie Disease. Genomics 1989, 4(4), 552-559. 5. Bach, A. W.; Lan, N. C.; Johnson, D. L.; Abell, C. W.; Bembenek, M. E.; Kwan, S. W.; Shih, J. C. cDNA Cloning of Human Liver Monoamine Oxidase A and B: Molecular Basis of Differences in Enzymatic Properties. Proc. Natl. Acad. Sci. U. S. A. 1988, 85(13), 4934-4938.


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Light-Up Probes Based on Fluorogens with Aggregation-Induced

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