Synthesis and characterization of styryl-BODIPY derivatives for monitoring in vitro Tau aggregation

Sensors and Actuators B 244 (2017) 673–683

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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb

Research paper

Synthesis and characterization of styryl-BODIPY derivatives for monitoring in vitro Tau aggregation Mani Vedamalai a , V. Guru Krishnakumar b , Sharad Gupta b , Shigeki Mori c , Iti Gupta a,∗ a b c

Department of Chemistry, Indian Institute of Technology Gandhinagar, Village Palaj, Simkheda, Gandhinagar, 382355, Gujarat, India Department of Biological Engineering, Indian Institute of Technology Gandhinagar, Village Palaj, Simkheda, Gandhinagar, 382355, Gujarat, India Integrated Centre for Sciences, Ehime University, Matsuyama, 790-8577, Japan

a r t i c l e

i n f o

Article history: Received 10 November 2016 Received in revised form 17 December 2016 Accepted 19 December 2016 Available online 3 January 2017 Keywords: BODIPY Fluorescence Tau Wolff-Kishner reduction

a b s t r a c t New synthetic strategy to synthesize ␣-methyl BODIPY derivatives from dipyrromethanes is reported. The method involves regioselective formylation of dipyrromethane followed by modified Wolff-Kishner reduction. The photophysical, electrochemical and computational studies of ␣-methyl BODIPY derivatives have been investigated in detail. The ␣-methyl BODIPY derivative was utilized further to prepare biologically important functionalized styryl-BODIPY library in high yield using microwave assisted Knoevenagel condensation. These synthesized dye derivatives were screened to find a potential candidate to track real-time in vitro tau protein fibrillization. Quinoxaline functionalized styryl-BODIPY derivative (5i) exhibited significant fluorescence enhancement upon binding to tau fibrils. Furthermore, tau-5i conjugate was systematically characterized by emission, aggregation kinetics, fluorescence microscopy and Atomic Force Microscopy techniques. Cell culture studies proved that compound 5i was cell permeable and non-toxic to live cells. In addition, a mechanism by which 5i interacts with tau fibrils has been elucidated which can be potentially exploited to further develop reporting dyes and inhibitors for tau aggregates. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Fluorescence sensing is an essential technique in modern scientific research due to its high sensitivity, real-time monitoring, greater spatial and temporal resolution [1,2]. Metal ions [3], highly reactive oxygen species [4,5], metabolites [6], amyloids [7], DNA [8] and cell signalling [9] have been successfully mapped in live systems using fluorescent probes. BODIPY dyes are commercially recognized fluorescent reporters for biological applications due to their excellent photophysical properties [10–12]. Most importantly, BODIPY allows many post-synthetic modifications on meso, ␣ and ˇ positions [13,14]. However, modification on meso aryl substituent does not have effect on absorption and emission bands as the BODIPY core and meso-aryl are orthogonally related [15]. Chemical modification on BODIPY skeleton is essential to shift the emission in near infra-red region for potential biomedical applications [16]. Various synthetic approaches such as transition metal catalyzed nucleophilic substitutions [17], condensation reaction with an aromatic aldehyde [18], Wittig reaction [19], aromatic

∗ Corresponding author. E-mail address: [email protected] (I. Gupta). http://dx.doi.org/10.1016/j.snb.2016.12.104 0925-4005/© 2017 Elsevier B.V. All rights reserved.

ring fusion [20], acid-mediated intra-molecular cyclization [21], annulation [22] and aza-substitution at meso position [23] have been utilized to shift the absorption and emission bands to red region. Metal catalyzed reactions need expensive catalysts and lack functional group tolerance while methyl substituted pyrroles are expensive. Difficulties in a conventional approach to synthesize ␣methyl(s) substituted BODIPY in high yield limit the application of BODIPY based probes. In order to overcome these setbacks, we have optimized the synthetic strategy to prepare ␣-methyl BODIPY derivatives from dipyrromethanes. Furthermore, ␣-methyl BODIPY was used to create a library of mono-styryl BODIPY derivatives in high yields for bio-applications. This strategy is versatile and can be used to prepare variety of ␣-methyl BODIPY derivatives from wide range of aromatic aldehydes. Functional aromatic group containing Styryl-BODIPYs have potential applications as bio-markers in life science. Neurodegenerative disorders such as Front Temporal Dementia (FTD), Alzheimer’s disease (AD), Parkinson’s disease (PD) and Huntington’s disease (HD) are marked by intracellular and extracellular deposits of proteins and peptides aggregates [24,25]. Monitoring the aggregation pathway of these proteins is crucial to understand the mechanism behind the formation of cytotoxic aggregates. In Alzheimer’s disease, abnormal tau forms Paired Helical Filament

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Scheme 1. Synthetic procedure for compounds 4a and 4b.

(PHFs) and Neurofibrillary Tangles (NFTs) which results in neuronal death, microtubule disassembly and impaired cargo traffic [26]. Therefore, tracking the tau fibrillation is a matter of prime importance to halt the aggregation using specific small molecules or peptide based inhibitors. There is an urgent need for a sensitive dye which can closely monitor the formation of protein aggregates. Single molecule fluorescence has been used to explore the migration and formation of insoluble tau aggregates in both intra and extra cellular environments [27]. To monitor the pathophysiology of NFT, fluorescent probes are ideally suited tool owing to high sensitivity, non-invasive nature and high temporal resolution. Noninvasive early detection of insoluble tau deposits will immensely help to cure the neurodegenerative diseases. Quinoline [28], benzimidazole [29], phenyldiazenyl benzothiazole [30], curcumin [31], pyrimidines and pyridazines [32] moieties have been successfully used to study tau pathology. It is very clear that nitrogen-based styryl heteroaromatic derivatives are effectively encapsulated into the binding pockets of tau fibrils. The modified approach reported herein will open up a new avenue to synthesize BODIPY based derivatives in high yields. The present study also examined the potential use of styryl-BODIPY derivatives as a marker to track real-time in-vitro aggregation of tau.

2. Results and discussion 2.1. Synthesis of 3-methyl BODIPY (4a), 3,5-dimethyl BODIPY (4b) and mono styryl-BODIPY derivatives (5a–j) Design and synthesis of 3-methyl BODIPY (4a) and 3,5-dimethyl BODIPY (4b) have been outlined in Scheme 1. First, anisyl dipyrromethane (1) had been prepared according to the reported straight forward method and details can be found elsewhere [33]. Mono ˛-formylated dipyrromethane (2a) was synthesized more selectively by the modified procedure. Compound 2a was obtained by treating compound 1 with an equivalent amount of benzoyl chloride in dry dimethyl formamide under nitrogen atmosphere. bis-formylated anisyl dipyrromethane (2b) was synthesized by subjecting compound 1 to phosphorous oxychloride in dry dichloromethane [34]. Compounds 2a and 2b were converted under modified Wolff-Kishner reduction to compounds 3a and 3b, respectively. During the reduction, excess hydrazine and water molecules were removed under reduced pressure. In next step, oxidation of compounds 3a and 3b was performed followed by addition of triethylamine and boron trifluoride dietherate with continuous stirring. The resulted BODIPY derivatives 4a and 4b were obtained in 72–74% yileds after neutral alumina column chromatography. The mono styryl-BODIPY derivatives (5a–j) were synthesized by microwave assisted Knoevenagel condensation of 4a as shown in Scheme 2.

The condensation reaction was achieved by continuous microwave irradiation of ␣-methyl BODIPY for 20 min at 110 ◦ C in ethanol. Functional aromatic aldehydes for the synthesis of compounds 5f–5h were synthesized by already reported synthetic procedures [35–37]. All mono styryl-BODIPY derivatives were synthesized from compound 4a and the yields were higher than those derived from 1,3,5,7-tetramethyl BODIPY precursor [38–40]. 4-fluorobenzyl (5a), anisyl (5c) and 1-pyrenyl (5j) substituted mono styrylated derivatives obtained in 81–89% yields. Naphthyl and bromo-carbazole functionalized styryl derivative (5e and 5f) were obtained in ∼73% yields. Tolyl and pyridyl aldehydes gave corresponding styryl products 5b and 5d in 60% yields. Aromatic amine based aldehydes produced blue colored desired derivatives (5h and 5g) in 89% yield. Quinoxaline functionalized mono-styryl-BODIPY derivative (5i) was obtained in 73% yield from 3-methyl-2-quinoxalinecarbaldehyde.

2.2. X-ray structure The X-ray crystal structures of 4a and 4b were solved, the ORTEP diagrams of both are shown in Fig. 1. The single crystals of compound 4a were developed by slow evaporation of the n-hexanes solution. The single crystals of compound 4b were obtained by slow evaporation of n-hexanes/dichloromethane solution. Compound 4a formed orange platelet crystals with P21/c (#14) space group while compound 4b gave red colored crystals with P21/n (#14) space group. Crystal data refinement parameters of compounds 4a and 4b have been provided in Table S1. The values of N1-B1-F1, N1-B1-N2, and F1-B1-F2 for the compound 4a were 128.84(16), 110.86(16) and 109.52(16), respectively and the results were comparable with typical BODIPY core [41]. In 4a the torsion angles between BODIPY plane and anisyl moiety [C6-C5-C11-C16] and [C4-C5-C11-C12] were 46.14(19) and 43.94(19) respectively; these values were relatively lower than the already reported meso-phenyl BODIPY[41] and meso-anisyl BODIPY [42]. Torsion angles in 4b between BODIPY core and anisyl moiety [C6-C5-C11-C16] and [C4-C5-C11-C12] were 57.41(16) and 55.44(15) respectively. The torsion angles in 4b were higher than those of 4a due to the symmetric functionalization at ˛-positions of BODIPY skeleton. C10-H10A bond length (of ␣-methyl group) was 0.98 A◦ which was higher than the typical C-H distance of ␣-methyl BODIPY core; suggesting highly acidic nature of methyl protons. Also, in 1 H NMR spectrum, 3-methyl protons in 4a appeared at ı 2.68 ppm while 3,5-methyl groups in 4b appeared at ı 2.65 ppm; these chemical shifts indicated more acidic nature of 3-methyl protons of 4a. 1,3,5,7-teramethyl BODIPY derivative exhibited shielded methyl protons signal at ı 2.55 ppm [43,44]; while the 1,3-dimethyl substituted BODIPY core showed 3-methyl protons at ı 2.59 ppm [45]. These results suggest that the increase of electron donating

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Scheme 2. Synthetic procedure for mono styryl-BODIPY (5a–j).

Table 1 Photophysical properties of BODIPY derivatives in methanol (a- ref [47]). Compounds

Absorption 1 ␭abs nm

Absorption 2 ␭abs nm

Shoulder peak ␭abs nm

Emission ␭em nm

Quantum yield ␾f

Stokes shift cm−1

4a 4b 5a 5b 5c 5d 5e 5f 5g 5h 5i 5j

498 505 555 561 569 552 566 590 598 599 569 595

376 374 314, 372 320, 410 335, 415 308, 379 375 365, 426 374 382, 433 346, 399 400

– – 524 528 536 502 502 557 499 – 533 –

511 518 565 574 589 562 590 629 640 680 579 635

0.110 0.252a 0.171 0.182 0.240 0.355 0.276 0.170 0.009 0.013 0.471 0.137

510.8 497.0 318.9 403.7 596.8 322.3 718.7 1050.9 1097.4 1988.6 303.5 1058.7

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Fig. 1. ORTEP diagrams of (a) 4a (CCDC 1442197); (b) 4b (CCDC 1468771). Thermal ellipsoids are shown at 50% probability level.

2.3. Photophysical properties

(a) 6

5x10

1,4-Dioxane 1-Butanol Acetone Acetonitrile Dichloromethane DMSO Ethanol Hexanes Methanol Tetrahydrofuran Toluene

6

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4x10

6

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6

2x10

6

1x10

0 575

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Intensity (a. u.)

(b) 1.25x10

5

1.00x10

5

7.50x10

4

5.00x10

4

2.50x10

4

Glycerol 0 % Glycerol 20 % Glycerol 40 % Glycerol 60 % Glycerol 80 % Glycerol 100 %

0.00 575

600

625

650

675

700

Wavelength (nm) Fig. 2. (a) Solvatochromism of compound 5i in different solvents at 20 ◦ C. (b) Emission spectra of compound 5i in mixtures of ethylene glycol (EG) and glycerol (GL) at 20 ◦ C. The excitation wavelength was 555 nm.

groups on BODIPY core reduces the acidic nature of methyl protons. Also, compound 4a had highly acidic protons and the acidity of ␣-methyl protons could be further fine-tuned by substituting appropriate meso-aryl groups.

Photophysical properties of all BODIPY derivatives were recorded in methanol (Table 1). Compound 4a showed absorption and emission bands at 498 and 511 nm respectively and the values were similar to typical BODIPY derivatives [18,46]. Compound 4b exhibited red-shifted absorption and emission bands with respect to compound 4a and values were 505 and 517 nm respectively. The presence of methyl group(s) at ␣-position of BODIPYs (4a and 4b) increased the fluorescence quantum yields significantly which were multiple times higher than the parent BODIPY [42]. The mono-styryl derivatives 5a–i exhibited weak and broad absorption band around 320–400 nm due to S0 -S2 (␲-␲*) transitions. An intense higher energy band at 400 nm was observed for 5j, on the other hand 5b and 5c exhibited weak absorption bands at 320 and 335 nm respectively. Compounds 5f, 5g, 5h and 5j showed absorption bands at 590, 598, 599 and 595 nm respectively. Multiple absorption peaks were observed for 5f due to the presence of multiple functional groups. All styryl-BODIPY derivatives had displayed a clear shoulder peak on high energy side except 5h and 5j. New absorption band observed for 5h at 433 nm due to N,N-bis(2-chloroethyl) benzenamine motif. Fluorescence quantum yield of BODIPY derivatives 5a to 5c was increasing in ascending order as electron density increases due to the presence of the electron donating group at para position. Hence, p-fluoro benzene appended BODIPY derivative (5a) had low fluorescence quantum yield than p-methoxy benzene appended BODIPY derivative (5c). Fluorescence quantum yield of compounds 5e and 5j were 0.276 and 0.137, respectively. Low fluorescence quantum yields were observed for p-amino aromatic derivatives (5f–5h), due to effective intra-molecular charge transfer (ICT) from aromatic amine to BODIPY core and molecular rotation of N–substituents. Compound 5 j exhibited emission band at 635 nm due to strong ␲ conjugation; while ICT probes 5f, 5h and 5g produced emission bands at 629, 680 and 640 nm respectively. Among the ICT probes, 5f had shown relatively high quantum yield because of the partially restricted molecular rotation by bulky carbazole moiety. The lower values of fluorescence quantum yield clearly indicated that non-radiative transitions were very high in 5g than 5h. Quinoxaline and pyridine substituted derivatives (5i and 5d) displayed high fluorescence quantum yield than other mono styryl derivatives. Large Stokes shifts were observed for 5g and 5h due to high molecular relax-

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Fig. 3. Illustrative presentation of the frontier molecular orbitals and the Kohn-Sham HOMO and LUMO energy levels of compounds 4a, 4b and TMB.

ation. Compounds 5a, 5d and 5i had small Stokes shift owing to the restricted intramolecular rotations. Particularly for 5i, the presence of 3-methyl group on quinoxaline moiety produced intense absorption band as well as caused restricted intra-molecular rotations which led to high quantum yield and low Stokes shift. The presence of electron donating anisyl group at meso-position of BODIPYs increased both the solubility and fluorescence quantum yield of the molecules; while emission band was progressively red shifted with increasing electron donating styryl substituents.

2.4. Solvatochromism Solvatochromic study of compound 5i was carried out in eleven different solvents ranging from polar to non-polar such as: 1,4-dioxane, 1-butanol, acetone, acetonitrile, dichloromethane, dimethyl sulfoxide, ethanol, methanol, tetrahydrofuran, hexane and toluene (Fig. 2a). The emission bands of 5i were 13 nm red shifted, starting from 578 to 591 nm upon increasing the solvent polarity as a result of solvent-solute interactions. In hexane, the wavelength of emission band maximum was at 578 nm while the emission intensity centered at 581 nm in acetone, acetonitrile, and methanol. The ethanolic solution of 5i exhibited emission maximum at 582 nm. Fluorescence emission maximum of compound 5i in 1-butanol and toluene appeared at 584 nm and the peak appeared at 585 nm in tetrahydrofuran and 1,4-dioxane. Dichloromethane and dimethyl sulfoxide had exhibited emission band at 586 and 591 nm, respectively.

Fig. 4. Expression and purification of recombinant tau protein (ht40) from E. coli cells. Purified tau protein was analysed by (a) SDS-PAGE stained with Coomassie blue R250 (b) Western blot immunolabelled with 5A6 antibody. Lane 1–Molecular weight marker, Lane 2–Tau protein (ht40). Molecular weights (kDa) of the standards are shown to the left of the images.

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40000

50000

(a)

Probe Probe + Tau fibrils

(b) 5i 5i+Tau fibrils

30000

Intensity (a. u.)

Intensity (a. u.)

40000

20000

10000

30000 20000

10000

0

0 5a

5b

5c

5d

5e

5f

5g

5h

5i

5j

Probes

575

600

625

Wavelength (nm)

Fig. 5. (a) Screening of Styryl-BODIPY derivatives (5 ␮M) as a probe towards tau aggregates (5 ␮M) in 20 mM of phosphate buffer (pH 7.4) containing 100 mM of NaCl and 1.25 ␮M of heparin. Data were collected at the wavelength of emission maximum of respective dyes after 24 h of incubation. (b) Fluorescence emission profile of 5i in the presence and the absence of tau aggregates. Excitation wavelength and slit widths were 555 nm and 1 nm, respectively.

Fig. 6. Heparin induced tau protein aggregation in 20 mM phosphate buffer (pH 7.4) at 37 ◦ C with ThT and 5i as reporter dyes. Data was collected every 6 h for a period of 2 days with fluorescence excitation: ␭440 nm for ThT and ␭555 nm for 5i; fluorescence emission: ␭480 nm for ThT and ␭585 nm for 5i. Data represents an average of readings from 3 wells. Error bars represent standard error.

2.5. Effect of viscosity Fluorescence measurements of compound 5i were done in ethylene glycol and glycerol mixtures of different viscosities (Fig. 2b). Emission intensity of compound 5i increased upon increasing the viscosity of the medium due to restricted molecular rotations in the highly viscous medium.

rized in Table S2. The larger separation between two reduction or oxidation peaks suggested that higher energy may require to achieve second reduction or oxidation in these molecules. These observations were comparable with typical green light emitting BODIPY derivatives [48]. Compound 4b showed more negative second reduction potentials as compared to 4a, which indicates that the addition of one more ␣-methyl group on BODIPY makes it difficult to reduce.

2.6. Electrochemical analysis 2.7. Computational calculations Cyclic voltammetry studies of compounds 4a and 4b were carried out in dry dichloromethane (DCM) at a platinum electrode using glassy carbon and saturated calomel electrode (SCE) as working and reference electrodes respectively. Compounds 4a and 4b showed two reversible reduction and oxidation waves in solution. The cyclic voltammograms of 4a and 4b are given in Fig. S1 (ESI); for compound 4a the separation values of the reduction and oxidation peaks were 1.022 and 0.666 V respectively. Similarly, the separation values of the reduction and oxidation peaks were 0.900 and 0.630 V respectively. The electrochemical results are summa-

The optimization of the molecular geometry of compounds 4a, 4b and TMB were done using density functional theory (DFT) calculations at B3LYP/6-31G+(d). The illustrative pictures and the Kohn-Sham HOMO and LUMO energy levels are presented in Fig. 3. Total energy and dipole moment of compound 4a were found to be −1066.325 a.u. and 6.3003 debye respectively. The calculated total energy and dipole moment of compound 4b were −1105.648 a.u. and 5.4229 debye respectively. In both the compounds, HOMO energy level was localized on the BODIPY

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Fig. 7. Fluorescence images of Tau fibrils. Images acquired after tau fibrillization (a) in presence of ThT (b) in presence of 5i. Images acquired after adding (c) ThT to pre-formed fibrils (d) 5i to pre-formed fibrils. ThT and 5i incubated together with pre-formed fibrils (e) excited using blue filter (f) excited using green filter (g) merged image of ThT and 5i fluorescence; white arrow shows the binding region of ThT and 5i to tau fibrils. (h) AFM image of tau fibril formed in presence of 5i after 48 h of aggregation. Distance between green arrow heads depicts the width of a tau fibril corresponding to 23 nm in diameter. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 8. Cell culture studies with compound 5i and HeLa cells. Internalization of compound 5i (100 nM) in live HeLa cells (a) Bright field image (b) fluorescence image (c) merged image (d) The viability of HeLa cells incubated with various concentration of compound 5i.

motif. In contrast to HOMO, LUMO was spreading on both BODIPY and phenyl motifs. The HOMO energy level of the compound 4b was slightly higher than that of compound 4a. According to DFT calculations, substitution of two methyl groups at ␣-positions effectively stabilize the BODIPY as compared to the mono ␣-methyl substituted BODIPY. The values of HOMO-LUMO energy gap for compounds 4a and 4b were −3.021 and −2.966 eV respectively. As the total energy reduces with a number of electrons donating methyl group on BODIPY skeleton, 3-methyl BODIPY derivative could be highly reactive than 1,3,5,7-tetramethyl BODIPY derivative (TMB). Frontier molecular orbitals of TMB were having high energy states than compounds 4a and 4b. Computational parameters for compounds 4a, 4b and TMB have been provided in Tables S3, S4 and S5 respectively.

2.8. BODIPY dyes as tau fibril binders An ideal tau aggregation reporter dye shall be selective towards the aggregated form i.e. it shall bind to oligomeric or fibrillar form but not to monomeric protein [49]. Also as the dye binds to the aggregated form, there shall be a detectable change in signal such as a shift in the absorbance peak or enhancement of the fluorescence emission signal or both [50]. To find dyes with these characteristics all styryl-BODIPY derivatives 5a–j were tested in vitro against aggregated form of tau.

Recombinant tau protein (ht40 isoform) was expressed in E. coli and subsequently purified by affinity chromatography to yield aggregation-assay ready tau protein with >98% purity as confirmed by gel electrophoresis and western blot (Fig. 4). Tau aggregation was carried out at 37 ◦ C in 20 mM phosphate buffer containing 100 mM NaCl, pH 7.4 at a protein concentration of 5 ␮M in the presence of individual dyes 5a–j. Under these conditions, in the presence of an anionic inducer such as heparin (1.25 ␮M), fibrillization process can be completed in 48 h. Hence, a midway time-point i.e. 24 h which was expected to have a significant population of all major forms of aggregates i.e. oligomers, protofibrils and fibrils were chosen for a preliminary screening. As shown in Fig. 5a, all the derivatives were virtually nonfluorescent in buffer due to strong non-radiative relaxation. When heparin induced tau aggregation was carried out in the presence of dyes separately, at 24 h 5i exhibited a very steep fluorescence enhancement of emission band at 585 nm (excitation wavelength 555 nm). In contrast, other mono styryl-BODIPY derivatives did not cause any significant fluorescence enhancement at respective wavelengths of emission maxima (Fig. 5a). Spectral properties of 5i in aggregate bound state were further investigated in scanning mode. A comparison with unbound dye showed that as 5i binds to tau aggregates, there was only a dramatic rise in emission intensity but no shift in the emission maxima (Fig. 5b). Since tau fibrillization (and formation of intermediates) involves conversion of native random coil to ␤-sheet structure, these results suggested that com-

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pound 5i selectively recognizes the ␤-sheet structures of various aggregated forms of tau than the hydrophobic patches on soluble monomers. Importantly, this study highlights some of the design features of fluorescent probes for ␤ sheets structures. ICT based fluorescent probes 5f, 5h and 5j did not show contrast emission signal upon incubation with tau aggregates individually. This indicated that aromatic amine could not bind with ␤-sheet of tau aggregates even though they are highly hydrophobic. Similarly, derivatives 5a, 5b, 5c, 5e and 5g did not cause any significant change in emission intensity as they lack polar functionality. Remarkably, pyridyl functionalized derivative 5d was also not able to bind to aggregated tau. The presence of 4-methoxy benzene and 2-methyl quinoxaline on 5i makes it a novel motif to bind with ␤-sheets structures of tau aggregates. The octanol/water partition coefficient of compound 5i was found to be 6, which confirmed that this derivative was highly lipophilic. 2.9. In vitro monitoring of tau fibrillization Progress of in vitro tau fibrillization is usually monitored by a non-interfering dye such as ThT (Thioflavin T) which upon binding to ordered structures comprising of stacked ␤-sheets exhibits several fold enhancement in fluorescence emission [51]. The presence of ThT does not affect the course of aggregation and it is highly selective towards fibrillary structures over amorphous aggregates, it is widely used for real-time monitoring of in vitro protein aggregation assays. Initial experiments suggested that 5i also exhibits enhanced fluorescence in the presence of aggregated tau similar to ThT. Before proceeding for ex vivo assay (Fig. 6) the dose response behavior of 5i was carefully investigated. The fluorescence intensity of 5i shows a dose response curve for tau (S2, SI). Accordingly, real-time tau aggregation was set up in a time dependent mode with respect to the changes in fluorescence intensity of ThT and 5i separately. For direct comparison, these experiments were carried out simultaneously in a 96-well plate using the same initial conditions with the only difference being the dye i.e. 5i or ThT. As shown in Fig. 6, a sigmoidal curve was obtained for tau aggregation kinetics with a distinct lag phase followed by an exponential phase and then a plateau for both ThT and 5i. Here, lag phase represented the time in which tau monomers come in close proximity using heparin as scaffold leading to nuclei or small oligomeric structure formation. This was followed by exponential phase in which larger oligomers and proto-fibrils were formed. A rapid increase in 5i fluorescence from baseline was observed indicating that similar to ThT, 5i also binds to stacked ␤-sheets structures of growing fibrils and does not interfere with the fibrillization process. For both dyes, emission attained a plateau ∼40 h of incubation as a result of the completion of fibrillization process. As both 5i and ThT displayed nearly overlapping curves, it can be inferred that both dyes bind to same structures during the course of aggregation. Overall, 8-fold increase in the fluorescence was observed for 5i upon incubation with tau as compared to dye only control without tau protein and qualitatively this enhancement was of the same order as observed for ThT incubated with tau. For dye only samples, a gradual decrease in signal was noticed for 5i which can be attributed to self-aggregation or photo-bleaching. 2.10. Imaging tau fibrils To further study the binding preferences of 5i towards tau aggregates post-fibrillization samples were further incubated for 2 additional days, thus allowing newly formed fibrils to mature and imaged by fluorescence microscopy. As shown in Fig. 7a and 7b, presence of fibrils with signature ghostly appearance was confirmed for aggregated tau samples formed in the presence of ThT as well as 5i. This confirmed that 5i does not interfere with the

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fibrillization process. Furthermore, similar to ThT, 5i was also incorporated into growing tau fibrils as indicated by the strong fluorescence signal. To further examine the preference for aggregated species and binding sites, tau protein was allowed to form fibrils under identical conditions sans dyes for 48 h and immediately stained with ThT and 5i separately. At this point most of the protein has assumed protofibrillar or fibrillar conformation and maturation process has started. As shown in Fig. 7c and d, ThT and 5i seem to bind to different regions of newly formed fibrils. In addition, 5i also appeared to stain nonfibrillar aggregates as well, as indicated by the appearance of red colored dots of varying sizes. This was in contrast to binding preferences obtained from images of aggregated tau formed in the presence of 5i and ThT separately (Fig. 7a and b). These result were very intriguing and to further understand the cause of this anomaly, post fibrillization staining experiment was repeated with equimolar concentration of both dyes being used simultaneously. Images were recorded for defined field of view by exciting ThT and 5i using respective filters (Figs. 7e and 7f) and then merged together as represented in Fig. 7g. A comparison of 7e with 7c and 7f with 7d indicated that there is no competition between ThT and 5i as far as binding sites on- and off-fibrils are concerned and both dyes stain different features. The merged image (Fig. 7g) revealed that 5i binds to terminal regions of the growing fibril, perhaps protofibrils and does not bind to entire length of the matured fibrils as evident by the capping of green fibrils (stained with ThT) with red dots (stained with 5i) indicated by white arrows. This also helps explain the differences observed with the staining properties of ThT and 5i with respect to tau fibrils formed in presence of dye against those stained post-fibrillization. Since 5i stains only the ends of the fibrils, it may get trapped in growing tau fibril when the aggregation is carried out in the presence of 5i, the end result being that the matured fibrils contain 5i throughout the length. On the other hand, ThT stains a fibril through intercalation post-fibrillization and does not appear to bind to the ends of the growing fibrils. These observation points to a novel binding mechanism for 5i that can be potentially exploited to further develop selective reporting dyes and inhibitors for tau aggregates. The surface morphology of tau fibrils formed in presence of 5i was further analysed by AFM (Fig. 7h) which appeared similar to fibrils formed in the presence of ThT with a uniform fibril width of 23 nm [52]. This confirmed that the incorporation of dye 5i does not perturb the morphology of tau fibrils. 2.11. Cell culture studies For any potential intracellular imaging applications, the fluorescent probe must exhibit cell permeability. In addition, live cell imaging is possible only if the probe does not exhibit toxicity towards cultured cells. Accordingly, 5i was tested for cellular uptake and cytotoxicity using HeLa cells. As shown in Fig. 8a-c, even at a very low concentration of 100 nM, strong fluorescence signal was observed in the cytoplasmic region of the live cells indicating that 5i is easily internalized. In addition, 5i did not cause any significant toxicity to cells for 24 h of treatment at all tested concentrations (5–40 ␮M) which was much higher than 100 nM used for cellular uptake studies (Fig. 8d). More than 95% cells were viable at the highest concentration of 40 ␮M. This suggested that the presence of 5i does not adversely affect the cellular processes and hence can be safely used for cell culture based studies including those involving live cell imaging. 3. Conclusion We have demonstrated a simple strategy to synthesize ␣methyl BODIPY derivatives and mono styryl-BODIPY library of

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biologically sensitive fluorescence probes consisting of various electron donor and acceptor motifs. The HOMO and LUMO energy levels of mono-methyl substituted BODIPY derivative were lower than those of dimethyl substituted BODIPY derivative’s frontier molecular orbitals. The real-time monitoring of tau aggregation which was at par with conventional dye ThT, demonstrated the usefulness of styryl-quinoxaline BODIPY derivative 5i as a reporter dye. Post-fibrillization staining studies have further revealed the binding preferences of 5i which points at a novel mechanism of binding previously not observed with ThT. Furthermore, the compound is cell permeable, non-toxic to live cells and thus can be developed for in vitro biological imaging application.

Acknowledgements IG thank SERB [Govt. of India, Grant No: EMR/2015/000779], SG thank SERB [Govt. of India, Grant No: EMR/2014/000336] and IIT Gandhinagar for financial support. IG is appreciative to Prof. H. Furuta, Kyushu University for X-ray analysis. MV is grateful to the IIT Gandhinagar for a post-doctoral fellowship. GK is thankful to MHRD for a doctoral fellowship. The authors would like to thank Lata Rani, IIT Gandhinagar for fruitful discussion on theoretical calculations.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2016.12.104.

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Biographies M. Vedamalai received his M.Sc., in Inorganic Chemistry from University of Madras and Ph.D. degree from National Chiao Tung University, Taiwan.H is research interests focus on developing fluorescent chemosensors for metal ions and biomolecules. V. Guru KrishnaKumar received M. Tech. degree from PSG College of Technology, Anna University. He is currently pursuing his PhD under the supervision of Prof. Sharad Gupta at Indian Institute of Technology Gandhinagar. His research interests include post translational modification mediated aggregation of the tau protein and tau derived amyloidogenic peptides involved in Alzheimer’s disease (AD), designing peptides and small molecules based inhibitor therapeutics for AD. Sharad Gupta obtained M.Sc. in Chemistry from Indian Institute of Technology Kanpur India and Ph.D. from University of Pittsburgh, USA. He performed postdoctoral research at Temple University, USA and then at University of Delaware, USA where he worked in the field of Neurodegenerative Diseases. He is Assistant Professor in Biological Engineering at Indian Institute of Technology Gandhinagar, where his early work deals with the development of novel probes for the detection of various conformations of aggregation prone proteins such as tau, A␤ and ␣-Synuclein. He is also engaged in research aimed at design and synthesis of peptides based aggregation inhibitors against tau protein. Shigeki Mori was born in Japan. He received his Ph.D. degree on the metal coordination of meso-aryl pentaphyrins and hexaphyrins from Kyoto University in 2008. After the postdoctoral research, he began an academic career in Ehime University, where he is currently a Lecturer. His research interests include design and application of porphyrin-related compounds. Iti Gupta obtained PhD in Chemistry from Indian Institute of Technology Bombay, India. She did postdoctoral research at Kyushu University, Japan where she worked on expanded porphyrins. Later she joined BITS-Pilani KK Birla Goa campus as faculty in Chemistry. Currently she is Associate Professor in Chemistry at Indian Institute of Technology Gandhinagar. Her research interests lie in design and synthesis of porphyrins and boron based fluorescent dyes for biological applications.