An Unconventional Mechanistic Insight on Aggregation Induced

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An Unconventional Mechanistic Insight on Aggregation Induced Emission in Novel Boron-Dipyrromethenes and their Rational Biological Realizations Roop Shikha Singh, Ashish Kumar, Sujay Mukhopadhyay, Gunjan Sharma, Biplob Koch, and Daya Shankar Pandey J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.6b05548 • Publication Date (Web): 10 Sep 2016 Downloaded from http://pubs.acs.org on September 12, 2016

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The Journal of Physical Chemistry

An Unconventional Mechanistic Insight on Aggregation Induced Emission in Novel Boron-Dipyrromethenes and their Rational Biological Realizations Roop Shikha Singha, Ashish Kumara, Sujay Mukhopadhyaya, Gunjan Sharmab, Biplob Kochb and Daya Shankar Pandeya* a

Department of Chemistry, Institute of Science, Banaras Hindu University, Varanasi - 221 005

(U.P.) India. b

Department of Zoology, Institute of Science, Banaras Hindu University, Varanasi - 221 005

(U.P.) India.

* To whom correspondence should be addressed. Email: [email protected], Phone + 91 542 6702480; FAX + 91 542 2368174

ACS Paragon Plus Environment


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ABSTRACT: Quinolone and quinoline based boron dipyrromethenes (BODIPYs) viz. BQN1 and BQN2 obtained by relative stabilization of keto and enol forms of N-methylated quinolones via minute substitutional variations (–H/−OCH3) have been reported. Relative disparity in degree of aromaticity arising from quinolone/-quinoline strongly affects the free rotation of these molecules. The photophysical and structural characteristics of these compounds revealed an exceptional dissonance between restriction of intramolecular rotation (RIR) and aggregation induced emission (AIE) signifying competitive steric hindrance and conjugation. Despite being an AIE inactive dye, BQN1 experiences maximum RIR and excels as a viscosity sensitive hindered molecular rotor, while an effective J-aggregation irrevocably established AIE in BQN2. This is the first report dealing with utilization of AIE active BODIPY (BQN2) in fabrication of AIEgen loaded bovine serum albumin (BSA) nanoparticles with live cell imaging in human breast cancer cell line MDA-MB-231. Binding mode of human serum albumin (HSA) to BQN2 has also been determined by molecular docking studies. In addition, viscochromism of BQN1 has been visualized through apoptotic marking in MDA-MB-231 cell line.


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INTRODUCTION Since the constructive utilization of aggregates through aggregation induced emission (AIE), organic luminophores have been enjoying a renaissance in the field of materials science and biology.1 The AIE materials exhibit wide architectural diversity ranging from hydrocarbons to heteroatom containing fluorophores and phosphorescent organometallic complexes.2−5 This diligent versatility has enabled us to understand the AIE mechanisms including restriction of intramolecular rotation (RIR), conformational planarization, E/Z isomerization, excimer/ J-aggregate formation, twisted intramolecular charge transfer (TICT), excited state intramolecular proton transfer (ESIPT), and conical intersection.1 Following these mechanistic insights it has been possible to decipher a structure-property relationship in AIE luminogens which enables their rational designing with widespread applications. Most of the reports on AIE feature RIR as prime factor for the phenomenon as evidenced by viscosity experiments.6−8 Such hindered molecular rotors have largely been investigated for mapping live cell viscosity and utilized in biomedical field as indicator for various diseases such as atherosclerosis, diabetes, and Alzheimer’s disease based on abnormal intracellular viscosities.9,10 Aromaticty and conjugation have a great influence on RIR and AIE. It has been established that AIE is weakened in more conjugated molecules due to unavailability of freely rotatable groups leading to competition between conjugation and RIR.11 Further, AIE has been recognized as an effective tool for fabricating polymer encapsulated AIE nanoparticles (NPs), as AIE luminogens defy self-quenching of the organic dyes making the NPs useful in cell and tissue imaging.12−16



On the other hand, BODIPYs represent a class of highly sought after molecules owing to their ease of synthesis and outstanding optical properties which enables them to act as labels for biomolecules. Versatile synthetic transformations in BODIPY dyes have raised a possibility for these to achieve high quantum yield even in solid state by using steric hindrance as an effective means to prevent close packing. This strategy has proved to be highly beneficial in establishing the potential of BODIPYs as cell imaging probes.17−26 AIE has also been introduced in BODIPYs via formation of J-aggregate, introduction of AIE active substituents and asymmetrization.27−29 However, appropriate strategies leading to AIE driven applications for BODIPYs, particularly in biomedical fields are still wanting.

ACQ

R=H

keto 3 BQN2

enol R = H, 1; OMe, 2

R = OMe

4

Decreased Free-rotation

BQN1

Increased Conjugation

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AIE

Figure 1. Substituent directed fabrication of BQN1 and BQN2. In this context, we have shifted our attention from quinolines30 and chose quinolone derivatives with an intention of altering aromaticity in BODIPY conjugates which may provide deep insights into conjugation vs rotation theory and thus designed BQN1 and BQN2 using –H and –OCH3 as pendants (Figure 1). Much to our surprise, the substituents played a crucial role in determining the end products through relative stabilization of keto and enol forms of quinolones,


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where –H substitution afforded quinolone (BQN1) while −OCH3 gave quinoline (BQN2) based BODIPYs. Through this contribution we describe substituent directed fabrication of two new BODIPYs BQN1 and BQN2 which challenge the interdependence of AIE and RIR through competitive steric hindrance and conjugation. Present work also contemplates the realization of discrete properties of BQN1 and BQN2 in biological system via fabrication of BQN2 loaded NPs and BQN1 mediated apoptotic cancer cell marking.

EXPERIMENTAL SECTION Reagents. The solvents were dried and distilled prior to their use following standard literature procedures.31 Pyrrole, trifluoroacetic acid, acetanilide, anisidine, iodomethane, 2,3dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), triethylamine and boron trifluoride diethyl etherate were procured from Sigma Aldrich India and used as received without further purifications. Aldehydes 2-oxo-1,2-dihydroquinoline-3-carbaldehyde (1) and 6-methoxy-2-oxo1,2-dihydroquinoline-3-carbaldehyde (2) have been prepared following the literature procedures.32 General Methods. Elemental analyses for C, H, and N were obtained on an Elementar Vario EL III Carlo Erba 1108 from microanalytical laboratory of the Sophisticated Analytical Instrumentation Facility (SAIF), Central Drug Research Institute (CDRI), Lucknow, India. Electronic absorption spectra were acquired on Shimadzu UV-1601 spectrophotometer. Fluorescence spectra (95% aqueous-methanol) at rt was acquired on a Perkin-Elmer LS 55 Fluorescence spectrometer. 1H, and 13C spectra were acquired on a JEOL AL 300 FT-NMR and JEOL resonance ECZ 500R using tetramethylsilane (Si(CH3)4) as an internal reference. Electrospray ionization mass spectrometric (ESI-MS) measurements were made on a Bruker



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Daltonics Amazon SL ion trap mass spectrometer. Samples were dissolved in 100% acetonitrile with 0.1% formic acid and introduced into the ESI source through a syringe pump at a flow rate of 100 mL/h. The capillary voltage was 4500 V, dry gas flow 8 L/min at 300 °C. The MS scan was acquired for 2.0 min and spectra print outs averaged of over each scan. SEM and TEM images were acquired on a JEOL JSM 840 A Scanning and EM-410 LS Transmission Electron Microscope, respectively. Fluorescence microscopic images were obtained on EVOS FL Cell Imaging System. Crystal data for BQN1 and BQN2 were collected on a dual source super nova CCD system from Agilent Technologies (Oxford Diffraction) at rt with Mo-Kα radiation (λ = 0.71073 Å). The structures were solved by direct methods (SHELXS 97) and refined by fullmatrix least squares on F2 (SHELX 97).33,34 Non-hydrogen atoms were refined with anisotropic thermal parameters. Hydrogen atoms were geometrically fixed and refined using a riding model. Computer program PLATON was used for analyzing the interaction and stacking distances.35,36 The CCDC deposition No 1455192−1455193 contains supplementary crystallographic data for this paper. Synthesis of 3. To a solution of aldehyde 1 (0.6 g, 3.47 mmol) in dimethylformamide (10 mL) K2CO3 (1.20 g, 8.68 mmol) was added. After stirring for 1 h it was treated with CH3I (1eq) and resulting reaction mixture was allowed to stir at r.t. for an additional 4 h. Following completion of reaction the contents of the flask were poured into ice cold water. Resulting precipitate was filtered, washed with water and dried under vacuum to give 3 as bright yellow solid. Yield: 0.371 g, 62%. Analytical data: Anal. Calc. for C11H9NO2: C, 70.58; H, 4.85; N, 7.48; Found C, 70.53; H, 4.83; N, 7.46%.1H NMR (DMSO-d6, 500 MHz, δ ppm): 3.62 (3H, s, −NCH3 proton), 7.31 (1H, t, aromatic-H), 7.56 (2H, d, J = 8.0 Hz, aromatic-H), 7.75 (1H, t, aromatic-H), 7.94 (1H, d, J = 8.0 Hz, aromatic-H), 8.45 (1H, s, methine-H), 10.23 (1H, s, −CHO


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proton). 13C NMR (DMSO-d6, 125 MHz, δ ppm): 29.7, 115.7, 119.3, 123.4, 125.2, 132.4, 134.7, 141.8, 142.3, 161.4, 190.6. Synthesis of 4. It was prepared following the above procedure for 3 using the aldehyde 2 (0.6 g, 2.95 mmol) in place of 1. Yield: 0.355 g, 59%. Analytical data: Anal. Calc. for C12H11NO3: C, 66.35; H, 5.10; N, 6.45; Found C, 66.32; H, 5.07; N, 6.40%. 1H NMR (DMSO-d6, 500 MHz, δ ppm): 3.65 (3H, s, −OCH3 proton), 3.72 (3H, s, −OCH3 proton), 7.20 (1H, t, aromatic-H), 7.31(2H, d, J = 8.5 Hz, aromatic-H), 7.61 (1H, t, aromatic-H), 7.84 (1H, t, aromatic-H), 10.21 (1H, s, −CHO proton). 13C NMR (DMSO-d6, 125 MHz, δ ppm): 29.6, 54.7, 55.9, 115.9, 118.6, 123.3, 126.0, 131.4, 134.2, 141.6, 143.1, 162.0, 190.3. Synthesis of 5. A round bottomed flask containing 3 (1.0 g, 5.34 mmol) was charged with pyrrole (10.0 mL) and catalytic amounts of trifluoroacetic acid (3 drops). The contents of the flask were stirred at r.t. for 24 h and after completion of the reaction (monitored by TLC) ensuing solution was concentrated to dryness under reduced pressure. Crude product thus obtained was purified by column chromatography (SiO2; ethylacetate/hexane). Off white colored band was collected and concentrated to afford the desired product. Yield: 0.742 g, 74%. Analytical data: Anal. Calc. for C19H17N3O: C, 75.23; H, 5.65; N, 13.85. Found: C, 75.19; H, 5.64; N, 13.81%. 1H NMR (CDCl3, 300 MHz, δ ppm): 3.70 (3H, s, −NCH3 proton), 5.60 (1H, s, meso-H), 5.99 (2H, s, pyrrolic-H), 6.12 (2H, s, pyrrolic-H), 6.69 (2H, s, pyrrolic-H), 7.26 (2H, m, aromatic-H), 7.53 (2H, t, aromatic-H), 7.66 (1H, s, methine-H), 9.10 (2H, br, pyrrolic –NH). 13

C NMR (CDCl3, 75 MHz, δ ppm): 30.0, 41.5, 106.4, 108.1, 108.2, 114.0, 117.1, 117.2, 120.6,

122.5, 128.7, 130.2, 131.3, 133.7, 137, 139.0, 162.7. Synthesis of 6. This compound was prepared by following the above procedure for 5 using aldehyde 4 (1.0 g, 4.60 mmol). Yield: 0.675 g, 68%. Analytical data: Anal. Calc. for



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C20H19N3O2: C, 72.04; H, 5.74; N, 12.60; Found C, 72.00; H, 5.69; N, 12.53%. 1H NMR (CDCl3, 300 MHz, δ ppm): 3.68 (3H, s, −OCH3a), 3.83 (3H, s, −OCH3b), 5.57 (1H, s, meso-H), 6.0 (2H, s, pyrrolic-H), 6.12 (2H, s, pyrrolic-H), 6.69 (1H, s, pyrrolic-H), 6.94 (1H, s, aromatic-H), 7.14 (1H, m, aromatic-H), 7.24 (1H, d, J = 7.2 Hz, aromatic-H), 7.6 (1H, s, aromatic-H), 9.17 (2H, br, pyrrolic –NH). 13C NMR (CDCl3, 75 MHz, δ ppm): 30.1, 55.6, 106.3, 108.1, 110.1, 115.3, 117.1, 119.2, 121.4, 131.4, 134.0, 136.6, 154.9. Synthesis of BQN1. DDQ (0.749 g, 3.3 mmol) dissolved in benzene (25.0 mL) was added drop wise over an hour to a stirring solution of 5 (1.0 g, 3.3 mmol) in dichloromethane (15.0 mL) and reaction mixture allowed to stir for an additional 2 h. After completion of the reaction contents of the flask were concentrated to dryness under reduced pressure. Resulting crude product was dissolved in dichloromethane (25.0 mL) and filtered to remove any solid impurities. The filtrate thus obtained was in-situ treated with BF3.Et2O (3.5 mL) in presence of triethylamine (0.75 mL) and reaction mixture stirred for 15 min at r.t.. Progress of the reaction was monitored by TLC and after completion the solution was filtered, filtrate washed thrice with water, extracted with dichloromethane, and concentrated to dryness under reduced pressure. Resulting crude product was charged on a flash column (SiO2; CH2Cl2/hexane). Dark orange-red band was collected and concentrated to dryness to afford the desired product. Yield: 0.279 g, 28%. Analytical data: Anal. Calc. for C19H14BF2N3O: C, 65.36; H, 4.04; N, 12.04; Found C, 65.29; H, 4.00; N, 12.01%. 1H NMR (CDCl3, 500 MHz, δ ppm): 3.82 (3H, s, −NCH3 proton), 6.51 (2H, s, pyrrolic-H), 6.99 (2H, s, pyrrolic-H), 7.34 (1H, t, aromatic-H), 7.47 (1H, d, J = 8.5 Hz, aromaticH), 7.65 (1H, d, J = 7.5 Hz, aromatic-H), 7.71 (1H, t, aromatic-H), 7.91 (3H, d, J = 6.0 Hz, merged signal for 2 pyrrolic and 1 aromatic proton). 13C NMR (CDCl3, 125 MHz, δ ppm): 30.3, 114.6, 118.6, 119.4, 123, 125.9, 129.8, 130.7, 132.4, 135.5, 140.7, 141.2, 141.6, 144.8, 160.3.


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Synthesis of BQN2. It was prepared following the above procedure for BQN1 using 6 (1.0 g, 3.0 mmol) in place of 5. Yield: 0.212 g, 21%. Analytical data: Anal. Calc. for C20H16BF2N3O2: C, 63.35; H, 4.25; N, 11.08; Found C, 63.31; H, 4.18; N, 11.03%. 1H NMR (CDCl3, 500 MHz, δ ppm): 3.93 (3H, s, −OCH3a), 3.99 (3H, s, −OCH3b), 6.51 (2H, s, pyrrolic-H), 6.83 (2H, s, pyrrolic-H), 7.09 (1H, s, aromatic-H), 7.41(1H, m, aromatic-H), 7.85 (1H, d, J = 9.0 Hz, aromatic-H), 7.94 (2H, s, pyrrolic-H), 7.98 (1H, s, aromatic-H). 13C NMR (CDCl3, 125 MHz, δ ppm): 27.8, 32.0, 53.9, 55.7, 106.3, 118.6, 123.2, 128.7, 130.9, 139.5, 144.6. Fabrication of BQN2 Loaded BSA-NPs. Ethanolic solution of BQN2 (1 wt%) was added dropwise to a solution of BSA (1 mg/mL) in phosphate buffer saline (PBS) where it formed nanoaggregates which are believed to interact with BSA. It was further sonicated in presence of glutaraldehyde which promoted cross linking of the BSA-matrix to produce spherical BQN2 loaded BSA-NPs. Molecular Docking. To ascertain possible binding sites for biomolecules, molecular docking studies on BQN2 have been performed using HEX 6.1 software and Q-site finder, which is an interactive molecular graphics program for interaction and docking calculations. DFT calculations were carried out using GAUSSIAN 09 by B3LYP methods. The geometries of BQN2 were optimized using standard 6-31G** basis set for C, H, N, O, and B.37 The coordinates for compounds were taken from their optimized structures as a .mol file and transformed to PDB format using CHIMERA 1.5.1 software. Visualization of the docked molecules has been made with Discovery Studio 3.5 software. By default, the parameters used for docking calculations were correlation type shape only, FFT mode at 3D level, and grid dimension 6 with receptor range 180 and ligand range 180 with twist range 360 and distance range 40. Cell imaging Experiment.



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Preparation of Stock Solution, Cell line and Cytotoxicity Assay. Stock solutions of BQN1 and BQN2 were prepared in dimethylsulfoxide (DMSO) and further diluted with complete DMEM (Dulbecco Modified Eagle Medium). Cytotoxicity of the compounds were checked on human breast cancer cells (MDA-MB-231) by 3-(4,5-dimethyl-thiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cytotoxicity of the BQN1 and BQN2 was evaluated on the basis of colour of formazan crystals formed by reaction of MTT and mitochondrial enzymes of the live cells. 1×104 cells/well were seeded in 96-well plate for 24 h and treated with of BQN1 and BQN2 (different concentrations) in DMEM with 10% fetal bovine serum (FBS) and incubated for 24 h. The medium was removed and MTT solution (10 µL/well) added from the stock (5 mg/mL) in PBS with 100 µL complete DMEM. The cells were further incubated for 2 h at 37 °C and resulting formazan crystals dissolved in DMSO (100 µL). The absorbance (at λ = 570 nm) was measured using an ELISA plate reader and results expressed as percentage of the cell viability with respect to control. Dose response curves were obtained by plotting percent cell survival (percent control) vs. compound concentrations. Fluorescence Imaging. 1× 105 MDA-MB-231 cells were seeded into 6 well plates and after 24 h incubation these were again incubated with BQN1 (50 µM ) and BQN2 (10 µM) in DMEM with 10% FBS for 1 h. Hereafter the cells were stained with Hoechst 33342 for nucleus staining and washed with PBS. Phase contrast and fluorescent images were captured by EVOS FL inverted fluorescence microscope (Life Technologies). Fluorescence Microscopy of Dead and Dying Cells with BQN1.1× 105 MDA-MB-231 cells with complete medium were seeded into 6 well tissue culture plates and incubated overnight for adherence. Cisplatin from Cipla (cisplatin injection BP Cytoplatin-50 Aqueous, each 50 mL vial contains: cisplatin IP 50 mg, sodium chloride IP 0.9% w/v and water for injection IP to 50 mL),


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15 µg/mL was added into each well and further incubated for 24h in 5% humidified CO2. The media was removed and cells incubated with BQN1 (10µM) for 1 h and after successive washing stained with Annexin V in HEPES buffer (10 mM, 137 mM NaCl, 3.2 mM KCl, pH 7.4). The cells were further incubated at 37 °C for another 30 min. The unbound stains were removed with HEPES buffer and imaging of the cells performed using inverted fluorescence microscope. Fluorescence Microscopy of BQN2 loaded BSA-NPs. 1× 105 MDA-MB-231 cells were seeded into 6 well plates and incubated for 24 h. The media was removed; BQN2 (10µM) and BQN2 loaded BSA-NPs (10 µM) were added to separate wells with complete DMEM for 1 h. Again compounds and media were removed and cells washed with PBS, and imaged by inverted fluorescence microscope.

RESULTS AND DISCUSSION Synthesis and Characterization of BQN1 and BQN2. Syntheses of the aldehydes 1 and 2 have been achieved following literature procedures.32 It was presumed that N-methylation of these aldehydes should give different products (keto/enol). N-methylation of the aldehydes has been performed by reacting these with CH3I in presence of a mild base K2CO3 (Scheme S1, Supporting Information). As expected, it happens to be the key step that determines the fate of aldehydes on the basis of relative stability of keto/ enol forms. As it has already been reported electron donating groups stabilize enol over keto forms, here too, 2 having an electron donating group (−OCH3) produced 4 while 1 with –H led to the creation of 3. The dipyrromethanes (5, 6) and BODIPYs (BQN1 and BQN2) have been synthesized using respective aldehydes following our earlier procedures.30 All the compounds have been thoroughly characterized by satisfactory elemental analyses, ESI-MS, 1H and

13

C NMR (Figure S1−S7, Supporting Information)



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spectroscopic studies. Molecular structures of both BQN1 and BQN2 have been authenticated by X-ray single crystal analyses (Figure S13, Supporting Information). Photophysical

Properties:

Aggregation

Induced

Emission/Aggregation

Caused

Quenching. The occurrence of AIE in BQN1 and BQN2 has been scrutinized by investigating their photophysical properties in solution (methanol) and aggregated (methanol-water) state. Noticeably, absorption profiles of BQN1 and BQN2 in methanol are almost similar. These displayed absorption maxima due to π−π∗ transitions at 507 and 503 nm while n−π* bands appeared in high energy side at 336 and 317 nm, respectively (Figure S8a, Supporting Information). Upon excitation at high energy wavelength these displayed dual emission at ~434 and 530 nm which have been ascribed to the emissions owing to quinolone/-quinoline and BODIPY units (Figure S8b, Supporting Information). Aggregation in these compounds has been followed in water-methanol mixture of varying water content. Absorption spectrum of BQN1 showed insignificant changes with increasing water fractions (fw) (Figure S9a, Supporting Information). On the other hand, upon increasing the water content from fw 10−90% its photoluminescence (PL) spectrum displayed continual decrease in the emission intensity for the bands due to quinolone (412 and 432 nm) and BODIPY (534 nm) moieties without apparent change in emission maxima. The red shifted emission band (~ 574 nm) at fw 100% signified formation of the aggregates however severe quenching in emission intensity of this band categorized BQN1 as an ‘aggregation caused quenching’ (ACQ) luminogen (Figure 2a). On the other hand, gradual increase in the water content to fw 100% diminished the absorbance for BQN2 at 503 nm with appearance of a red shifted band at 534 nm (Figure S9b, Supporting Information). This bathochromically shifted band represents a typical J-


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band indicating formation of J-type aggregates arising from slipped face-to-face packing between monomers (vide-infra).38

Figure 2. Emission spectra of BQN1 (a) and BQN2 (b) in THF/water mixture with different water fractions (c = 5 × 10−5mol/L). In its PL spectra, BQN2 exhibited considerable emission quenching up to fw 60% while further increase in water content (fw 70%) led to a red shifted band at 534 nm. Further increase in fw to 80% led to emergence of two new bands at 567 and 614 nm which attained maxima at fw 90%. A small decrease in emission intensity observed at fw 100% may be attributed to decreased solubility (Figure 2b). As well, fluorescence life time enhanced from 1.10 (methanol) to 3.89 ns (fw 90%) affirming formation of the aggregates in BQN2 (Figure S10, Supporting Information).1 In solution (methanol), BQN2 shows weaker emission due to active intramolecular rotations which promote the decay of excited state via non-radiative processes thus the molecule shows a lower fluorescence life time. With increasing water content molecules tend to form aggregates which restrict the intramolecular rotation and prevent non-radiative decay and molecule stays longer in excited state.



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Aforesaid experiments undoubtedly established AIE in BQN2 and ACQ in BQN1. The augmentation of AIE lies in mechanistic interpretation of this enticing phenomenon. As RIR is considered to be the most popular route for AIE, careful assessment of differential effect of intramolecular motion on emission behavior of respective molecules is highly desirable. The effect of viscosity on fluorescence is mainly governed by intramolecular motion, therefore emission spectra of BQN1 and BQN2 have been acquired in methanol-glycerol mixture with varying glycerol fractions (fg). Interestingly, emission of BQN1 rejuvenated to a greater extent (~30 times) at fg 100% while BQN2 showed insignificant variation with increasing fg (Figure 3a). In addition, time-resolved emission decay profile for BQN1 with increasing fg also revealed radical increase in fluorescence life time with an increase in viscosity strengthening the assumption of an effective RIR in BQN1 (Figure 3b).


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Figure 3. (a) Emission spectra of BQN1 in methanol/glycerol mixture and (b) fluorescence decays for BQN1 in methanol/glycerol mixture with different glycerol volume fractions (c = 5 × 10−5mol/L). Chemical structure of these molecules in Figure 1 shows greater conjugation in BQN2 due to presence of quinoline unit as compared to BQN1 having quinolone unit at meso- position. Enhanced conjugation in BQN2 lowers the rotational motion in methanol thereby enhancing fluorescence in solution as compared to BQN1. This observation indicated that RIR will be more effective upon increasing viscosity in BQN1 wherein effective conjugation is lowered due to presence of the quinolone unit. It is well known that rotation and conjugation cannot go hand in hand in AIE luminogens,16 in sharp contrast BQN1 experiencing greater RIR comes out as an ACQ dye while despite of being more conjugated, BQN2 is AIE active. Crystal Packing Patterns. From the fluorescence spectra of these molecules it is apparent that both BQN1 and BQN2 form aggregates in methanol-water mixture therefore the aggregates should be responsible for emission quenching and enhancement in these molecules. To ascertain the mechanism of aggregate build-up single-crystal X-ray structures of these molecules have been determined (Figure S13, Supporting Information) and supramolecular packing modes thoroughly analyzed. The packing in BQN1 revealed parallel boraindacene units interacting in a head-to-head manner with adjacent ones through single π−π interaction (Figure 4a).



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(a) (b)

(c)

(d)

Figure 4. Crystal packing in BQN1 via π−π interaction (a) Packing in BQN2 via π−π interactions in a herring-bone mode (b) Helical structure of BQN2 through π−π interactions (c) and packing of BQN2 via C-H···π interactions (d). It displayed strong overlap between quinolone units which are 68.11° out of the boraindacene plane, while a very small overlap between boraindacene planes due to horizontal slipping. The shortest carbon-carbon distance between boraindacene planes is 3.361 Å. Clearly, BQN1 forms a one-dimensional chain which is distantly related to J-aggregate. Crystal packing in BQN2 undoubtedly revealed that molecules stack through π−π interactions in a herringbone mode, typical for J-aggregates with a long range ordered structure (Figure 4b). Head-to-tail


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arrangement of the adjacent quinoline and boraindacene planes in a slipped manner create a helical structure (Figure 4c). This helix is further strengthened by two C−Η···π interactions (Figure 4d). A closer look in the packing of BQN2 revealed no overlap between adjacent quinoline and boraindacene units with shortest carbon-carbon distance between adjacent quinoline units being 3.372 Å, which is slightly longer than BQN1. Completely slipped packing and minimum interaction between the adjacent units in BQN2 justifies stronger and effective Jaggregation in BQN2 relative to BQN1. Aggregation Induced Emission vs. Restriction of Intramolecular Rotation.

Unusual

relationships between the RIR and AIE in these compounds can be rationalized by considering competition between steric hindrance and conjugation. RIR is not much effective in BQN2 due to increased conjugation relative to BQN1 and at the same time it should restrain the AIE attribute too. However, crystal packing pattern shows a typical J-aggregate with slipped stacking and without any overlap in BQN2, which may be created from steric hindrance offered by – OCH3 in close vicinity to boraindacene plane. Thus, the steric effect prevails upon conjugation strength in BQN2 and causes AIE. In BQN1, despite the presence of a less conjugated structure and more potent RIR effect emission quenching is observed due to close crystal packing. Morphological Analysis of Nanoaggregates of BQN2.

SEM and TEM analyses of

nanoaggregate suspension of BQN2 obtained at fw 90% showed branched and reticulated nanofibers (Figure 5). As suggested by SEM, the nanofibers displayed a broad size distribution with their thickness ranging from 25−250 nm. DLS study has also been performed on nanofibers of BQN2 which showed their average size to be 181 nm (Figure S12a, Supporting Information). In earlier work, we have categorically shown that intermolecular interactions play a decisive role in creating morphological variation through relative orientation of the hydrophilic and



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hydrophobic domains.30 The same can be realized in BQN2 too as aforesaid interactions led to fibrous aggregate by bringing the hydrophilic –OCH3 and BF2 units at the periphery and hydrophobic quinoline at the centre of helix. Distinct properties of BQN1 and BQN2 may be realized through potential application of these molecules in living systems.

a

b

c

Figure 5. SEM images at 2000X (a) and 10000X (b) magnification and TEM (c) images of the nanofibers of BQN2 formed in methanol/water (fw 90%) mixture (c = 5 × 10−5mol/L). Live Cell Imaging Experiments. Cellular imaging experiments have been carried out on human breast cancer cell line (MDA-MB-231). Non-toxicity of the compounds under investigation toward biological systems has been verified by MTT assay (Figure S14, Supporting Information). Visualization of Viscochromism in Living Cells during Apoptosis. As envisaged by viscochromism BQN1 behaves as a fluorescent molecular rotor and attains emission only after free rotations are hindered. Considering biocompatibility of the quinolones, BODIPY dyes and viscosity enhancement during apoptosis an attempt has been made to visualize viscosity changes in apoptotic cancer cells.39,40 In this direction, apoptosis has been induced in the cells by 24 h incubation of cisplatin followed by exposure to PS-binding protein Annexin V (covalently


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labeled with green fluorescent dye, Alexa Fluor488) (Supporting Information).41 Another set of healthy cells were placed in a separate well as control. The cisplatin induced apoptotic cells showed green fluorescence in presence of Annexin V (Figure 6) while healthy cells remained unaffected. Noticeably, healthy cells faintly stained upon treatment with BQN1 (Figure S15, Supporting Information), whereas apoptotic cells showed significant red emission. Yellow color in the overlay of microscopic images confirmed selective staining of the apoptotic cells by BQN1. These experiments strongly supported visualization of increased viscosity during apoptosis in MDA-MB-231 cells by BQN1.

Figure 6. Fluorescence micrographs of the control and dead/-dying MDA-MB-231 cells stained with Annexin V−Alexa Fluor488 and 10 µM of BQN1 (Annexin V−Alexa Fluor488 = green; BQN1 = red; bright field = gray). The dead and dying cells were treated with cisplatin (15 µM) for 24 h and incubated with 10 µM of BQN1 for 1 h at 37 °C and washed with HEPES buffer.



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Fabrication, Characterization and Live Cell Imaging of BQN2 Loaded BSA-NPs. As BQN2 behaves as an amphiphile with well defined hydrophilic (–OCH3 and BF2) and hydrophobic (quinoline) domains along with an AIE attribute, it may find potential application in fabrication of fluorophore loaded NPs.16 Bovine serum albumin (BSA) has been chosen as polymer matrix due to its cost effectiveness, biocompatiblity, biodegradablity, nontoxicity, nonimmunogenicity and its structural homology (76%) to human serum albumin (HSA).42,43 Owing to these properties BSA-NPs also serve as good candidates for tumor targeting as well as drug and antigen delivery.44-46 Keeping these in mind BODIPY (BQN2) loaded BSA-NPs have been fabricated for the first time and their efficacy in live cell imaging has been evaluated. Drop-wise addition of ethanolic BQN2 (1 wt%) to a solution of BSA (1 mg/mL) in phosphate buffer saline (PBS) led to the formation of nanoagregates which are believed to interact with BSA.47,48 Possible initial interactions have been followed by SEM which revealed interaction of the BSANPs with reticulated nanofibers of BQN2 creating a structure like dew on leaves (Figure 7a). It was further sonicated in presence of glutaraldehyde which promoted cross linking of the BSAmatrix to produce spherical BQN2 loaded BSA-NPs. Expectedly, fluorophore loaded BSA-NPs with spherical shape and an average diameter of 90 nm have been achieved and visualized by SEM (Figure 7b). Size of the NPs is in good agreement with those obtained from DLS measurements (100 nm) (Figure S12b, Supporting Information). It is worth mentioning that at very high fluorophore loading (> 5 wt%) it formed spheres of average diameter 400 nm, however these lacked uniformity in size distribution. Fluorescence microscopic phase contrast images showed knitted network of BQN2 and BSA as well as green and red emission of BQN2 loaded BSA-NPs under blue/green excitation (Figure S11c−f, Supporting Information).


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a

b

c

50µm

Figure 7. SEM image showing initial interaction of BQN2 nanofibers with BSA at 500x (a) and 4000x magnification and BQN2 loaded BSA-NPs (c). The absorption maximum of BQN2 (503 nm) blue shifted as compared to the BQN2 loaded BSA-NPs which displayed the maximum at 509 nm (Figure 8a). In its emission spectra the aggregates of BQN2 displayed emission at 567 and 614 nm whereas BQN2 loaded BSA-NPs produced a broad emission band at 540 nm with enhanced intensity which lies in between the emission peaks of BQN2 in methanol (534 nm) and its aggregate (Figure 8b). On the other hand, emission peak in nanofibers of BQN2 at 614 nm remained intact in BQN2 loaded BSA-NPs but its intensity significantly increased. A closer look at emission behavior of BQN2 loaded BSANPs revealed that these sustain native emission properties of BQN2 nanofibers.



Figure 8. Absorption (a) and emission (b) spectra of BQN2 (c = 5 × 10−5mol/L) in PBS buffer with different concentration of BSA (0−0.1 wt%).

GFP Channel

RFP Channel

GFP/RFP Overlay

100 µm

100 µm

100 µm

100 µm

100 µm

100 µm

100 µm

100 µm

BQN2

Phase Contrast

BQN2 loaded BSA nanoparticles

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Figure 9. Phase contrast and fluorescence micrographs of MDA-MB-231 cells stained with 10 µM of BQN2 (top) and enhanced fluorescence with more granular dots after exposure to BQN2 loaded BSA-NPs (bottom). Considering chronological exploitation of fluorescent NPs in bio-imaging we intended to utilize BQN2 loaded BSA-NPs in cell imaging and performed live cell imaging experiments on MDA-MB-231 cells (Supporting Information). BQN2 offered dichromic fluorescence of moderate intensity in MDA-MB-231 cells with selective cytoplasm staining (Figure S15, Supporting Information). Interestingly, after 5h incubation with BQN2 loaded BSA-NPs an intense dichromic fluorescence was easily visualized in these cells. The fascinating BQN2 loaded BSA-NPs retained its spherical shape in the cells and appeared as tiny dots with dichromic emission under blue or green excitation (Figure 9). Intense fluorescence of BQN2


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loaded BSA-NPs relative to its nanofibers also suggested increased uptake of BSA encapsulated NPs inside the cells leading to cogent realization of ‘enhanced permeability and retention’ (EPR) effect.42 Molecular Docking with HSA. Observed shifts in the absorption and emission maxima of nanofibers of BQN2 upon interaction with BSA prompted us to investigate the most probable binding mode of this compound with HSA through molecular docking studies. HSA has been chosen over BSA for their structural homology and its relevance in human physiology. Further, it is believed that amphiphilic behavior of BQN2 and HSA may enable them to interact through both hydrophobic as well as H-bond interactions (Figure 10a).

(a)

(b)

Figure 10. (a) Docked model of BQN2 (stick) showing the binding with HSA (cartoon) (PDB ID: 1h9z) and (b) the interaction mode between BQN2 and HSA. Molecular docking studies revealed that hydrophobic quinoline unit gets inserted into the cavity, while hydrophilic BF2 unit interacts with the surface of HSA through H-bonds (Figure S16, Supporting Information). It has been observed that LEU115 forms two H-bonds, one with F-atom (1.643 Å) and other with N-atom (2.313 Å) of BODIPY core (Figure 10b). Further both polar/charged (ARG114, ARG117, ARG145, ARG186) and hydrophobic (PRO113, LEU115,



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LEU182, ILE142, MET123) amino acid residues are within 4 Å of BQN2 enabling the Hbonding, van der Waals, electrostatic and hydrophobic interactions. Possibility of these interactions can also be realized through absorption and emission spectra of BQN2 in presence of BSA (vide-supra). Nanofibers of BQN2 formed in PBS displayed a red shifted band as compared to BQN2, disappeared upon binding with BSA (Figure 8a) which further suggested the energy changes associated with binding of BSA to BQN2. In its emission spectra nanofibers of BQN2 displayed two bands at 567 and 614 nm among these the band at 567 nm blue shifted to 540 nm (Figure 8b) suggesting energy changes associated with H-bonding interaction of nanofibers with BSA. On the other hand, upon binding with BSA the band at 614 displayed emission enhancement without any shift of the peak position suggesting some sort of hydrophobic interactions, also. It is clear from the docking studies that binding between HSA and BQN2 is largely due to H-bonding interactions with small contribution from hydrophobic ones. These findings further strengthen our earlier assumptions related to absence of an effective RIR in BQN2 and dominance of intermolecular interactions (J-aggregation) in governing the emission enhancement processes.

CONCLUSIONS Summarily, sovereignty of quinoline-BODIPY conjugates in the area of AIE has been established by synthesizing novel BODIPYs BQN1 and BQN2 and determining the crucial role of substituents (−H, −OCH3) in quinoline-quinolone transformation via relative stabilization of enol over keto forms. In our quest to rationalize AIE we came across with the mutual incompatibility of AIE and RIR as BQN1 is AIE inactive, but bestowed with a persuasive RIR whereas BQN2 is AIE active with pronounced J-aggregation. The aforesaid discordance has


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been related to competitive steric hindrance and RIR adduced by varying aromaticity of quinolone (BQN1) and quinoline (BQN2). The BQN2 nanofibers have been encapsulated with BSA matrix to give AIEgen loaded BSA-NPs and its efficacy has been evaluated by live cell imaging in cancer cells. Binding mode of HSA with BQN2 has also been determined by molecular docking studies which further articulated the absence of an effective RIR in BQN2. Considering increased viscosity during cell death, viscochromism of BQN1 has been visualized through apoptotic marking in cancer cells. This study repudiates the conventions of AIE and presents a new insight on mechanistic interpretation which can be utilized for various applications, essentially driven by specific structural features.

ASSOCIATED CONTENT Supporting Information: CCDC Nos. 1455192−1455193 (BQN1−BQN2) also contain supplementary crystallographic data for this paper. 1H and

13

C NMR spectra, ESI-MS, UV−vis

and fluorescence spectra, details of live cell imaging experiments and Tables S1−S2 (PDF) Crystallographic data for BQN1−BQN2 (CIF). This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Authors E-mail: [email protected]. Acknowledgements



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We acknowledge financial support from the Department of Science and Technology (DST), New Delhi through the Scheme SR/S1/IC-25/2011. R. S. Singh acknowledges the University Grants Commission, New Delhi, India for a Senior Research Fellowship (19-12/2010(i) EU-IV). REFERENCES 1. Mei, J.; Leung, N. L. C.; Kwok, R. T. K.; Lam, J. W. Y.; Tang, B. Z. Aggregation-Induced Emission: Together We Shine, United We Soar! Chem. Rev. 2015, 115, 11718−11940. 2. Niu, C.; You, Y.; Zhao, L.; He, D.; Na, N.; Ouyang, J. Solvatochromism, Reversible Chromism and Self-Assembly Effects of Heteroatom-Assisted Aggregation-Induced Enhanced Emission (AIEE) Compounds. Chem. Eur. J. 2015, 21, 13983–13990. 3. Chang, Z, -F.; Jing, L. –M.; Wei, C.; Dong, Y. –P.; Ye, Y. –C.; Zhao, Y. S.; Wang, J. –L. Hexaphenylbenzene-Based,

π-Conjugated

Snowflake-Shaped

Luminophores:

Tunable

Aggregation-Induced Emission Effect and Piezofluorochromism Chem. Eur. J. 2015, 21, 8504−8510. 4. Xie, Z.; Chen, C.; Xu, S.; Li, J.; Zhang, Y.; Liu, S.; Xu, J.; Chi, Z. White-Light Emission Strategy of a Single Organic Compound with Aggregation-Induced Emission and Delayed Fluorescence Properties. Angew. Chem. Int. Ed. 2015, 54, 7181 –7184. 5. Evans, D. A.; Lee, L. M.; –Baca, I. V.; Cowley, A. H. Aggregation-Induced Emission of Bis(imino)acenaphthene Zinc Complexes: Photophysical Tuning via Methylation of the Flanking Aryl Substituents. Organometallics 2015, 34, 2422–2428. 6. Guan, W.; Zhou, W.; Lu, C.; Tang, B. Z. Synthesis and Design of Aggregation-Induced Emission Surfactants: Direct Observation of Micelle Transitions and Microemulsion Droplets. Angew. Chem. Int. Ed. 2015, 54, 15160–15164.


Page 27 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


7. Ju, M.; Yuning, H.; Lam, J. W. Y.; Anjun, Q.; Youhong, T.; Zhong, T. B. AggregationInduced Emission: The Whole Is More Brilliant than the Parts. Adv. Mater. 2014, 26, 5429– 5479. 8. Leung, N. L. C.; Xie, N.; Yuan, W.; Liu, Y.; Wu, Q.; Peng, Q.; Miao, Q.; Lam, J. W. Y.; Tang, B. Z. Restriction of Intramolecular Motions: The General Mechanism behind AggregationInduced Emission. Chem. Eur. J. 2014, 20, 15349–15353. 9. Stutts, M.; Canessa, C.; Olsen, J.; Hamrick, M.; Cohn, J.; Rossier, B.; Boucher, R. CFTR as a cAMP-Dependent Regulator of Sodium Channels. Science 1995, 269, 847–850. 10. Luby-Phelps, K. Cytoarchitecture and Physical Properties of Cytoplasm: Volume, Viscosity, Diffusion, Intracellular Surface Area. Int. Rev. Cytol. 2000, 192, 189– 221. 11. Chen, B.; Nie, H.; Lu, P.; Zhou, J.; Qin, A.; Qiu, H.; Zhao, Z.; Tang, B. Z. Conjugation Versus Rotation: Good Conjugation Weakens the Aggregation-Induced Emission Effect of Siloles, Chem. Commun. 2014, 50, 4500−4503. 12. Reisch, A.; Klymchenko, A. S. Fluorescent Polymer Nanoparticles Based on Dyes: Seeking Brighter Tools for Bioimaging. Small 2016, 12, 1968−1992. 13. Zhu, Z.; Qian, J.; Zhao, X.; Qin, W.; Hu, R.; Zhang, H.; Li, D.; Xu, Z.; Tang, B. Z.; He, S. Stable and Size-Tunable Aggregation-Induced Emission Nanoparticles Encapsulated with Nanographene Oxide and Applications in Three-Photon Fluorescence Bioimaging, ACS Nano 2016, 10, 588−597 14. Wang, X.; Song, P.; Peng, L.; Tong, A.; Xiang, Y. Aggregation-Induced Emission Luminogen-Embedded Silica Nanoparticles Containing DNA Aptamers for Targeted Cell Imaging. ACS Appl. Mater. Interfaces 2016, 8, 609–616.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 35

15. Lia, K.; Liu, B. Polymer-Encapsulated Organic Nanoparticles for Fluorescence and Photoacoustic Imaging. Chem. Soc. Rev. 2014, 43, 6570−6597. 16. Qin, W.; Ding, D.; Liu, J.; Yuan, W. Z.; Hu, Y.; Liu, B.; Tang, B. Z. Biocompatible Nanoparticles with Aggregation-Induced Emission Characteristics as Far-Red/Near-Infrared Fluorescent Bioprobes for In Vitro and In Vivo Imaging Applications. Adv. Funct. Mater. 2012, 22, 771–779. 17. Kowada, T.; Maeda, H.; Kikuchi, K.; BODIPY-Based Probes for the Fluorescence Imaging of Biomolecules in Living Cells. Chem. Soc. Rev. 2015, 44, 4953−4972. 18. Murale, D. P.; Hong, S. C.; Yun, J.; Yoon, C. N.; Lee, J. –S. Rational Design of a PhotoCrosslinking BODIPY for In Situ Protein Labeling. Chem. Commun. 2015, 51, 6643−6646. 19. Brizet, B.; Goncalves, V.; Bernhard, C.; Harvey, P. D.; Denat, F.; Goze, C. DMAP-BODIPY Alkynes: A Convenient Tool for Labeling Biomolecules for Bimodal PET–Optical Imaging. Chem. Eur. J. 2014, 20, 12933–12944. 20. Ulrich, G.; Ziessel, R.; Harriman, A. The Chemistry of Fluorescent Bodipy Dyes: Versatility Unsurpassed. Angew. Chem., Int. Ed. 2008, 47, 1184–1201. 21. Ziessel, R.; Ulrich, G.; Harriman, A. The Chemistry of Bodipy: A New El Dorado for Fluorescence Tools. New J. Chem. 2007, 31, 496–501. 22. Loudet, A.; Burgess, K. BODIPY Dyes and Their Derivatives: Syntheses and Spectroscopic Properties. Chem. Rev. 2007, 107, 4891−4932. 23. Manzano, H.; Esnal, I.; -Matesanz, T. M.; Bañuelos, J.; -Arbeloa, I. L.; Ortiz, M. J.; Cerdán, L.; Costela, A.; -Moreno, I. G.; Chiara, J. L. Unprecedented J-Aggregated Dyes in Pure Organic Solvents. Adv. Funct. Mater. 2016, 26, 2756–2769.


Page 29 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


24. Lu, H.; Wang, Q.; Gai, L.; Li, Z.; Deng, Y.; Xiao, X.; Lai, G.; Shen, Z. Tuning the SolidState Luminescence of BODIPY Derivatives with Bulky Arylsilyl Groups: Synthesis and Spectroscopic Properties. Chem. Eur. J. 2012, 18, 7852−7861. 25. Ni, Y.; Lee, S.; Son, M.; Aratani, N.; Ishida, M.; Samanta, A.; Yamada, H.; Chang, Y. –T.; Furuta, H.; Kim, D.; Wu, J. A Diradical Approach towards BODIPY-Based Dyes with Intense Near-Infrared Absorption around λ=1100 nm. Angew. Chem. Int. Ed. 2016, 128, 2865−2869. 26. Dent, M. R.; López-Duarte, I.; Dickson, C. J.; Geoghegan, N. D.; Cooper, J. M.; Gould, I. R. Krams, R.; Bull, J. A.; Brooks, N. J.; Kuimova, M. K. Imaging Phase Separation in Model Lipid Membranes through the Use of BODIPY Based Molecular Rotors. Phys. Chem. Chem. Phys. 2015, 17, 18393−18402. 27. Choi, S.; Bouffard, J.; Kim, Y. Aggregation-Induced Emission Enhancement of a MesoTrifluoromethyl BODIPY via J-aggregation. Chem. Sci. 2014, 5, 751–755. 28. Şen, E.; Meral, K.; Atılgan, S. From Dark to Light to Fluorescence Resonance Energy Transfer

(FRET):

Polarity-Sensitive

Aggregation-Induced

Emission

(AIE)-Active

Tetraphenylethene-Fused BODIPY Dyes with a Very Large Pseudo-Stokes Shift. Chem. Eur. J. 2016, 22, 736–745. 29. Shimizu, S.; Murayama, A.; Haruyama, T.; Iino, T.; Mori, S.; Furuta, H.; Kobayashi, N. Benzo[c,d]indole-Containing

Aza-BODIPY

Dyes:

Asymmetrization-Induced

Solid-State

Emission and Aggregation-Induced Emission Enhancement as New Properties of a Well-Known Chromophore. Chem. Eur. J. 2015, 21, 12996–13003. 30. Singh, R. S.; Gupta, R. K.; Paitandi, R. P.; Dubey, M.; Sharma, G.; Koch, B.; Pandey, D. S. Morphological Tuning via Structural Modulations in AIE Luminogens With the Minimum



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 35

Number of Possible Variables and Their Use in Live Cell Imaging. Chem. Commun. 2015, 51, 9125−9128. 31. Perrin, D. D.; Armango, W. L. F.; Perrin, D. R. Purification of Laboratory Chemicals; Pergamon: Oxford, U.K., 1986. 32. Mukhopadhyay, S.; Gupta, R. K.; Biswas, A.; Kumar, A.; Dubey, M.; Hundal, M. S.; Pandey, D. S.; A Dual-Responsive “Turn-On” Bifunctional Receptor: A Chemosensor for Fe3+ and Chemodosimeter for Hg2+. Dalton Trans. 2015, 44, 7118–7122. 33. Sheldrick, G. M. SHELXL-97, Program for X-ray Crystal Structure Refinement; Gottingen University: Gottingen, Germany, 1997. 34. Sheldrick, G. M. SHELXS-97, Program for X-ray Crystal Structure Solution; Gottingen University: Gottingen, Germany, 1997. 35. Spek, A. L. PLATON, A Multipurpose Crystallographic Tools Utrecht University, Utrecht, The Netherlands, 2000. 36. Spek, A. L. ActaCrystallogr. A 1990, 46, C31. 37. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; et al. Gaussian 09; Gaussian, Inc., Wallingford, CT, 2009. 38. Kim, S.; Fujitsuka, M.; Tohnai, N.; Tachikawa, T.; Hisaki, I.; Miyata, M.; Majima, T. The Unprecedented J-aggregate Formation of Rhodamine Moieties Induced by 9-Phenylanthracenyl Substitution. Chem. Commun. 2015, 51, 11580−11583. 39. Zhu, H.; Fan, J.; Li, M.; Cao, J.; Wang, J.; Peng, X. A “Distorted-BODIPY”-based Fluorescent Probe for Imaging of Cellular Viscosity in Live Cells. Chem. Eur. J. 2014, 20, 4691–4696.


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40. Raut, S.; Kimball, J.; Fudala, R.; Doan, H.; Maliwal, B.; Sabnis, N.; Lacko, A.; Gryczynski, I.; Dzyuba, S. V.; Gryczynski, Z. A Homodimeric BODIPY Rotor as a Fluorescent Viscosity Sensor for Membrane-Mimicking and Cellular Environments. Phys. Chem. Chem. Phys. 2014, 16, 27037−27042. 41. Vermes, I.; Haanen, C.; Steffens-Nakken, H.; Reutellingsperger, C. A Novel Assay for Apoptosis Flow Cytometric Detection of Phosphatidylserine Early Apoptotic Cells Using Fluorescein Labelled Expression on Annexin V. J. Immunol. Methods 1995, 184, 39−51. 42. Tanaka, T.; Shiramoto, S.; Miyashita, M.; Fujishima, Y.; Kaneo, Y.; Tumor Targeting Based on the Effect of Enhanced Permeability and Retention (EPR) and the Mechanism of ReceptorMediated Endocytosis (RME). Int. J. Pharm. 2004, 277, 39−61. 43. Bolel, P.; Datta, S.; Mahapatra, N.; Halder, M. Spectroscopic Investigation of the Effect of Salt on Binding of Tartrazine with Two Homologous Serum Albumins: Quantification by Tartrazine with Two Homologous Serum Albumins: Quantification by Use of the Debye− Hü Ckel Limiting Law and Observation of Enthalpy−Entropy Compensation. J. Phys. Chem. B 2012, 116, 10195−10204 44. Zhou, Z. M.; Anselmo, A. C.; Mitragotri, S. Synthesis of Protein-Based, Rod-Shaped Particles from Spherical Templates using Layer-by-Layer Assembly. Adv. Mater. 2013, 25, 2723–2727. 45. Rodrigues, N. F.; van Tilburg Bernardes, E.; Rocha, R. P.; da Costa, L. C.; Coutinho, A. C.; dos Santos Muniz, M.; Pereira, A. A.; da Silva, P. H.; Malaquias, L. C.; Coelho, L. F. Bovine Serum Albumin Nanoparticle Vaccine Reduces Lung Pathology Induced by Live Pseudomonas Aeruginosa Infection in Mice. Vaccine 2013, 31, 5062–5066.



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46. Elzoghby, A. O.; Samy, W. M.; Elgindy, N. A. Albumin-Based Nanoparticles as Potential Controlled Release Drug Delivery Systems. J Control Release 2012, 157, 168–182. 47. Zhong, R.; Liu, Y.; Zhang, P.; Liu, J.; Zhao, G.; Zhang, F. Discrete Nanoparticle-BSA Conjugates Manipulated by Hydrophobic Interaction. ACS Appl. Mater. Interfaces 2014, 6, 19465−19470. 48. Li, W.; Chen, D.; Wang, H.; Luo, S.; Dong, L.; Zhang, Y.; Shi, J.; Tong, B.; Dong, Y. Quantitation of Albumin in Serum Using “Turn-on” Fluorescent Probe with AggregationEnhanced Emission Characteristics. ACS Appl. Mater. Interfaces 2015, 7, 26094−26100.


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Graphical Abstract



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199x142mm (150 x 150 DPI)


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210x118mm (150 x 150 DPI)


An Unconventional Mechanistic Insight on Aggregation Induced

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