Exploration of Molecular Shape-Dependent Luminescence Behavior

Subscriber access provided by EDINBURGH UNIVERSITY LIBRARY | @ http://www.lib.ed.ac.uk

Article

Exploration of Molecular Shape-Dependent Luminescence Behavior: Fluorogenic Organic Nanoparticles Based on Bent Shaped ESIPT Dyes Mithun Santra, Yong Woong Jun, Ye Jin Reo, Sourav Sarkar, Wanuk Choi, Ji Eon Kwon, Soo Young Park, and Kyo Han Ahn ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00040 • Publication Date (Web): 19 Jun 2018 Downloaded from http://pubs.acs.org on June 20, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 26 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

ACS Applied Bio Materials

Exploration of Molecular Shape-Dependent Luminescence Behavior: Fluorogenic Organic Nanoparticles Based on Bent Shaped ESIPT Dyes Mithun Santra,a Yong Woong Jun,a Ye Jin Reo,a Sourav Sarkar,a Wanuk Choi,b Ji Eon Kwon,c Soo Young Park*,c and Kyo Han Ahn*,a aDepartment

of Chemistry, Pohang University of Science and Technology (POSTECH), 77 Cheongam-Ro, Nam-Gu, Pohang,

Gyungbuk 37673, Republic of Korea. bDepartment

of Environmental Science and Engineering, Pohang University of Science and Technology (POSTECH) 77

Cheongam-Ro, Nam-Gu, Pohang, Gyungbuk 37673, Republic of Korea. cCenter

for Supramolecular Optoelectronic Materials, Research Institute of Advanced Materials (RIAM), Department of Materials

Science and Engineering, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea.

KEYWORDS: molecular shape-dependent luminescence, solid-state luminescence, fluorescent organic nanoparticles, excited-state intramolecular proton-transfer, fluorescence imaging.

ABSTRACT: Fluorogenic materials that emit both in solution and in solid states have received special attention for their potential applicability to optoelectronic devices and bio-chemical sensing systems. To develop such materials, we have explored the molecular shape-dependent luminescence behavior for the bent-shaped molecules. We have synthesized several bent-shaped 1(benzo[d]thiazol-2-yl)-6-substituted-naphthalen-2-ol compounds, which also have an excited-state intramolecular proton-transfer (ESIPT) feature, which is expected to modulate their optical properties. All the compounds and their 2-methoxy derivatives showed luminescence both in solution as well as in solid states, plausibly due to unfavorable orbital interactions between stacked molecules nearby as suggested by single crystal structure packing pattern analysis. The naphthol compounds also showed large Stokes shifts due to the ESIPT feature, along with good optical brightness and tunable emission color by changing the 6-substituent. Fluorescent organic nanoparticles were then prepared from selected compounds, and their size distribution and polydispersity were analyzed by dynamic light scattering and transmission electron microscope. Photophysical properties of the new dyes and their nanoparticles in solution as well as in solid states (in powder and crystalline forms) were characterized by spectral analysis and fluorescence life-time measurements. The new organic nanoparticles were shown to stain cells by both confocal

ACS Paragon Plus Environment

ACS Applied Bio Materials 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

and two-photon microscopic imaging. The approach of molecular shape control demonstrated here thus opens door toward solid-state luminescent organic compounds and their nanoparticles.

1. INTRODUCTION Fluorescent organic nanoparticles (FONPs) have received increasing attention for their potential applications to optoelectronic nanodevices, bio-chemical sensing and imaging, and drug delivery.1−13 Conversion of organic fluorophores into the corresponding nanoparticles may accompany changes in their optical properties and biocompatibility.14−18 FONPs generated from small fluorescent molecules have gained special interest, as they can be readily synthesized and derivatized, and also more biodegradadable.19−24 Such FONPs have potential for the detection and imaging of biological analytes.25−29. Most of the FONPs are based-on aggregation-induced emission (AIE) dyes; hence, they are non-emissive in solution but emissive upon aggregation mainly due to the conformational restriction. Recently, we have disclosed a different mechanism for the enhanced luminescence upon aggregation, that is, “non-resonant” stacking interaction or unfavorable exciton coupling of dyes due to unfavorable molecular shape.30 A dye molecule with bent shape can have the unfavorable excited state resonance interaction with nearby molecules and thus can emit luminescence in the aggregated states even though it has a parallel stacking pattern (Figure 1a) as in the case of the resonant dimer (H-aggregation). As the origin of luminescence of such a class of dyes is not due to the conformational restriction, they can be emissive not only in solid states but also in solution, a distinct feature from the AIE dyes (Figure 1b).


Page 2 of 26

Page 3 of 26 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


Figure 1. (a) Exciton energy diagram for the resonant (H-aggregation) and “non-resonant” dimers with parallel transition dipoles (Reproduced from reference 30. Copyright 2014 American Chemical Society). (b) A difference between “fluorescence-on” due to aggregation-induced emission and fluorescence change

due to the non-resonant stacking interaction (NRSI) upon aggregation.

In this contribution, we have explored the molecular shape-dependent luminescence behavior by investigating solid-state luminescence properties of bent-shaped 1-(benzo[d]thiazol-2-yl)-6substituted-naphthalen-2-ol (BTN) compounds and their 2-methoxy derivatives. The naphthol compounds also have an additional structure motif for the excited-state intramolecular protontransfer (ESIPT) (Figure 2). We expected that the bent shape of the molecules would endow them with luminescent behavior both in solution and in solid states, and the ESIPT feature would further modulate their photophysical properties. Fluorescent organic compounds that exhibit ESIPT states have attracted much attention for their potential applications in molecular switches, laser dyes, and UV photostabilizers, molecular probes and imaging agents.31−37 We have found that the emission wavelengths of the BTN dyes can be also tuned by changing the 6-substituent (Figure 2).



Page 4 of 26

To our delight, the ESIPT dyes and their 2-methoxy derivatives showed fluorescence in solid states, highlighting the usefulness of the molecular shape control approach for the development of solidstate luminescent materials. Accordingly, fluorescent nanoparticles were also generated by precipitation of the dyes in aqueous media. A handful number of ESIPT-based FONPs have been developed, but all of them are based on the AIE/AIEE phenomenon.38−42 The AIE/AIEE based dyes or their FONPs are non- or very weakly-emissive in solution but become more emissive upon aggregation, due to the conformational restriction mechanism mostly. In contrast, the BTN dyes are emissive in solid states as well as in solution. This strategy opens up a new perspective for the design and synthesis of organic dyes that are emissive not only in solution but also in solid states. Such dyes are readily transformed into the corresponding FONPs. We further show that the BTN dyes and their FONPs are potentially useful for fluorescence imaging of cells under one- as well as two-photon excitation conditions.

-Me S

ESIPT

N 1

S

OMe 2 3

N OH

R 6 5 4 BTN-OMe

R

NH O

R BTN

CN-BTN -OMe

R = CN

CN-BTN

Br-BTN -OMe

R = Br

Br-BTN

Pyr-BTN -OMe

S

R=

N

Pyr-BTN

Figure 2. Structures of the BTN dyes with different 6-substituents (R) with and their solid-state fluorescence images obtained under excitation at 365 nm.

2. RESULT AND DISCUSSION 2.1. Synthesis and Structural Characterization. The designed compounds were synthesized following the synthetic procedure as shown in Scheme 1. Starting from compound 1, compounds 2


Page 5 of 26 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


and 3 were synthesized through a substitution reaction with copper cyanide and a copper/L-prolinecatalyzed aryl amination reaction, respectively.43,44 Next, condensation reactions of aldehydes 1–3 with 2-aminothiophenol in refluxing acetonitrile in the presence of 5% (v/v) acetic acid for 12–16 h produced the corresponding benzothiazoles in good yields: CN-BTN-OMe (87%), Br-BTN-OMe (66%) and Pyr-BTN-OMe (65%). Finally, demethylation of these compounds with 1dodecanethiol and NaOH in N-methyl-2-pyrrolidone (NMP) at 130 °C for 2–3 h afforded the corresponding naphthols in good yields: CN-BTN (92%), Br-BTN (88%) and Pyr-BTN (83%). During the isolation stage of the products, we observed that all the compounds showed bright luminescence in the amorphous solid state, as shown by the photos taken under UV irradiation (Figure 2). The 6-cyano derivatives, CN-BTN-OMe and CN-BTN, emitted blue and cyan fluorescence, respectively, when irradiated with a UV lamp at 365 nm. The 6′-bromo-substituted dyes, Br-BTNOMe and Br-BTN, emitted blue and green fluorescence, respectively. The pyrrolidine analogues, Pyr-BTN-OMe and Pyr-BTN, emitted green and orange fluorescence, respectively. The emission band of BTNs shifted to the longer wavelength as the better electron-donor was substituted at the 6-position of the naphthalene ring. Given that BTNs emit in the solid state, we prepared the corresponding nanoparticles from three selected compounds (CN-BTN, Br-BTN and Pyr-BTN) by a precipitation method. In a typical procedure, each dye in DMSO (5 mM, 100 μL) was quickly dropped into deionized water (10 mL) under vigorous sonication at 40 °C. After being sonicated for additional 5 min, the solution was cooled to and kept at room temperature for 30 min. The solution contained colloidal nanoparticles in uniform dimensions as evidenced by DLS analysis. The FONPs had quite different hydrodynamic diameters and polydispersity index values (HDD, PDI): NPs of CN-BTN (45.4 nm, 0.34), NPs of Br-BTN (22.7 nm, 0.11), and NPs of Pyr-BTN (141.8 nm, 0.059). An analysis by transmission electron microscopy (TEM) for the NPs of Br-BTN showed well-defined and monodispersed nanospheres with an average particle size of 20 nm (Figure 3d).



S OMe

2: a

OMe

3: b

CN-NBT-OMe: R = CN (87%) OMe Br-NBT-OMe: R = Br (66%)

c

Pyr-NBT-OMe: R =

R

R 1

N

CHO

CHO

Br

Page 6 of 26

N

(65%)

2: R = CN 3: R =

d

N

S

N

CN-NBT: R = CN (92%) OH

Br

Br-NBT: R = Br (88%) Pyr-NBT: R =

N

(83%)

Scheme 1. Synthesis of the benzthiazolyl-naphthol compounds and their 2-methoxy derivatives. Reagent and conditions: (a) CuCN, N-methyl-2-pyrrolidone (NMP), 135 °C, 10 h; (b) CuI, K2CO3, L-proline, pyrrolidine, DMSO, 100 °C, 12 h; (c) 2-aminothiophenol, CH3CO2H, CH3CN, reflux, 12−16 h; (d) nC12H25SH, NaOH, NMP, 130 °C, 2−3 h.

Figure 3. DLS and PDI data for the BTN NPs: (a) CN-BTN, (b) Br-BTN and (c) Pyr-BTN. (d) TEM image of Br-BTN NPs. (e) Fluorescence images of the nanoparticles of CN-BTN, Pyr-BTN and PyrBTN-OMe in cell culture media DMEM, taken under irradiation at 365 nm using a UV lamp.


Page 7 of 26 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


2.2. Photophysical Properties in Solution. Absorption and emission spectra for all the dyes were recorded in different organic solvents, from nonpolar 1,4-dioxane to polar DMSO and phosphate buffer solution (PBS buffer pH 7.4). Photophysical properties of these dyes revealed that both the absorption and emission properties of them are dependent on the 6-substituent, as noted above. Compared to the cyano-substituted dyes, the amino-substituted ones showed bathochromic shifts both in the absorption and emission bands, in accordance of the color changes observed. Moreover, the 2-methoxy and 2-hydroxy-derivatives had absorption and emission bands in different regions, as the latter compounds can generate ESIPT tautomers (the keto forms) that emit at the longer wavelengths compared to the phenol tautomers (Figure 4). A striking difference in the emission intensity dependent on solvent was observed between the hydroxyl and methoxy derivatives: for example, the BTN-OMe dyes emitted strongly regardless of the media, both organic and aqueous media, but the BTN analogues emitted strongly in DMSO, moderately in EtOH, but poorly in other solvents (dioxane, dichloromethane, acetonitrile, and PBS buffer). This contrasting emission behavior can be explained by the dyes’ ESIPT tendency that is dependent on media. It is known that the ESIPT process in a structurally related hydroxyphenyl– benzothiazole system (HBT) is promoted by the intramolecular hydrogen bonding between the hydroxyl hydrogen and the benzothiazole nitrogen. In a solvent that destabilizes the intramolecular hydrogen bond in the HBT dyes, the ESIPT is suppressed.45−47 The polar aprotic solvent DMSO destabilizes the intramolecular hydrogen bonding in the BTN dyes, which precludes the ESIPT process. In such a case, the dye can emit from the normal localized excited (LE) state: In other words, BTN dyes in DMSO behave as the corresponding BTN-OMe. In ethanol, the ESIPT occurs partially, hence the BTN dyes emit moderately. In the other organic solvents, the ESIPT occurs favorably, and the BTN dyes emit through the keto form with very lower quantum efficiency as usual (Фfl = 0.005 in the case of HBT).48 CN-BTN-OMe emitted in all the organic solvents examined and also in PBS buffer when excited at 340 nm, and its absorption and the emission wavelength maxima showed a bathochromic shift with increasing the solvent polarity from 410 to 430 nm (Figure S1). In contrast, CN-BTN had a rather broad absorption band in the range of 350– 450 nm depending on the solvent. Similar to the case of the methoxy analogue, the emission also showed a red-shift from 410 to 490 nm upon increasing the solvent polarity (Figure S2c).



CN-BTN-OMe CN-BTN 0.10

Br-BTN-OMe Br-BTN Pyr-BTN-OMe

0.05

0.00 350

Pyr-BTN

400 450 500 Wavelength (nm)

550

(b)

CN-BTN-OMe

Normalized Fluorescence

(a)

Absorbance

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 8 of 26

CN-BTN 0.9

Br-BTN-OMe Br-BTN

0.6

Pyr-BTN-OMe Pyr-BTN

0.3

0.0 400

500

600

700

Wavelength (nm)

Figure 4. (a) Absorption spectra, (b) normalized emission spectra of all the BTN dyes; all experiments were performed for 10 μM dye in EtOH (1% DMSO).

The bromo-substituted compounds also showed similar behavior: Br-BTN-OMe emitted in all the organic solvents as well as in PBS buffer with absorption maxima at 350 nm (Figure S3). In contrast, Br-BTN showed three absorption maxima at 320, 390 and 470 nm (Figure S4). Strong electron-donating pyrrolidine-substituted BTNs showed bathochromic shifts both in the absorption and emission wavelengths from the other fluorophores: Pyr-BTN-OMe showed an absorption maximum at 410 nm and fluoresces in all the organic and PBS buffer solution (Figure S5). The emission was red-shifted with increasing the solvent polarity, except in PBS buffer solution (Figure S5c). Pyr-BTN absorbed in the wavelength range of 400–500 nm and showed strong emission at 540 nm in DMSO that gave a homogenous solution of the dye (Figure S6). This dye showed a little red-shift in the emission with increasing the solvent polarity, and in PBS buffer, the emission came from the aggregated nanoparticles at 560 nm (Figure S6c). In pure water, PyrBTN (10 μM) also existed as nanoparticles due to aggregation. The bathochromic shifts in the emission wavelengths of all the dyes with increasing solvent polarity indicate that they have intramolecular charge-transfer (ICT) excited states in polar media.


Page 9 of 26 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


Table 1. Photophysical properties of CN-BTN, Br-BTN, Pyr-BTN and Pyr-BTN-OMe.

Dyea

λabs (nm)

CN-BTN

ФFf

Brightness (ε × ФF)e

Stokes Shifte (nm)

ФFσ2 (GM)g

0.373

0.076

6400

87, 111

n.d.h

0.001

0.084

0.001

800

70, 95

n.d.h

540

0.015

0.411

0.008

2000

130

2.2

532

0.234

0.307

0.033

1900

127

2.0

εb

λemc (nm)

ФFd

ФFe

373

17000

460, 484

0.031

Br-BTN

400

9700

470, 495

Pyr-BTN

410

4800

Pyr-BTNOMe

405

6000

o

All the measurements were conducted at 25 C with each of the compounds at 10 μM in DMSO. bLM−1cm−1. c Measured under excitation at the maximum absorption wavelength for each compound in DMSO. d-fThe fluorescence quantum yields (dEtOH, eDMSO, and fnanoparticles) in PBS buffer (pH 7.4), determined using fluorescein (ΦF = 0.79 in 0.1 M NaOH) and 9,10-diphenylanthracene (ΦF = 0.90 in cyclohexane) as reference dyes. gTwo-photon action cross section (TPACS) measured for each compound dissolved in DMSO (50 μM) using rhodamine B as a standard. n.d.h = not determined. a

The fluorescence quantum yields, optical brightness as well as Stokes shifts for selected dyes (CNBTN, Br-BTN, Pyr-BTN, and Pyr-BTN-OMe) were determined in EtOH and DMSO, respectively, along with their NPs in PBS buffer (pH 7.4) (Table 1). Interestingly, the BTN series showed significantly higher quantum yields in DMSO but relatively lower quantum yields in EtOH. Their NPs also showed low quantum yields in the buffer. For example, Pyr-BTN has significantly higher quantum efficiency in DMSO (ΦF = 41%) compared to that in ethanol (ΦF = 1.5%) and its NPs in the buffer (ΦF = 0.8%). CN-BTN showed a similar trend. Br-BTN showed lower quantum efficiency than the other two. In contrast to the BTN series, its methoxy-derivative Pyr-BTN-OMe showed good quantum efficiency both in EtOH and in DMSO (ΦF = 23% and 30%, respectively), although its NPs in the buffer had still low quantum yield (ΦF = 3%). A drop in the quantum yield in going from aprotic solvent DMSO to a protic solvent EtOH in the case of the BTN dye but not in the case of its methoxy derivative suggests that hydrogen bonding interaction provides a nonradiative decay route for the BTN dye. The molar absorptivity (ε) determined in DMSO decreased by changing substituent from the electron withdrawing group to the electron donating group. Thus CN-BTN has maximum absorptivity (ε = ~2 × 104) among all the fluorophores. The molar absorptivity of Br-BTN (ε = 9 × 103) is higher than those of Pyr-BTN-OMe and Pyr-BTN (ε = 6 × 103, and ~5× 103 respectively). The optical brightness (ε × ФF) of all these fluorophores decreased by changing the substituents from cyano to pyrrolidine, except for Br-BTN which had



Page 10 of 26

poor optical brightness due to weak quantum efficiency in DMSO. The maximum Stokes shifts were observed for Pyr-BTN-OMe and Pyr-BTN.

2.3. Solid-State Photophysical Properties. We have investigated solid-state (in the powder form) fluorescence properties of the selected fluorophores (CN-BTN, Br-BTN, Pyr-BTN, and PyrBTN-OMe). As displayed in Figure 5a and Table 2, there are significant bathochromic shifts in their emission bands as the 6-substituent changes from electron-withdrawing to –donating groups, as observed in the case of their molecular solutions. The maximum emission wavelengths are 498 nm, 541 nm, and 585 nm for CN-BTN, Br-BTN, and Pyr-BTN, respectively. Computational calculations indicate that the HOMO–LUMO energy gap is highest for CN-BTN (3.79 eV), followed by Br-BTN (3.73 eV) and Pyr-BTN (3.15 eV), which corroborates well with the absorption and emission behavior of the dyes in solution as well as in solid state (Figure S7). We also obtained emission spectra of Pyr-BTN and Pyr-BTN-OMe in their crystalline states (Figure 5c). The spectral data overlap very well with those obtained in their powder states (Figure 5a), which indicate that these dyes have essentially the same packing patterns in both powder and crystalline states. A large red shift (~ 70 nm) in the emission maxima between Pyr-BTN-OMe and Pyr-BTN, observed both in the powder and in the crystalline states, is due to the ESIPT process in the case of the BTN dye that makes it emit in the keto tautomer. The nanoparticles of CN-BTN, Pyr-BTN-OMe and Pyr-BTN in aqueous solution show bright blue, green and orange luminescence, respectively, under irradiation with a UV lamp at 365 nm (Figure 3e). The photophysical properties of those four representative compounds (CN-BTN, Br-BTN, PyrBTN, and Pyr-BTN-OMe) were also measured in their powder states, and the results are summarized in Table 2. All the compounds in their powder states show bathochromic shifts (Δλmax = 14–45 nm) in their emission maxima from those observed in DMSO. Pyr-BTN showed the largest shift and emits at 585 nm in the powder state. Their photoluminescence quantum (ФF) yields in the powder state show a similar trend to that observed in their nanoparticle forms in PBS buffer: CN-BTN (14.3%) > Pyr-BTN-OMe (13.9%) > Pyr-BTN (6.1%) >> Br-BTN (0.1%). The ФF yields measured in the crystalline state of Pyr-BTN-OMe (2.2%) is lower than that in the powder state, whereas Pyr-BTN (7.3%) shows similar ФF yields both in the crystalline and in the powder states (Table S1). The poor quantum efficiency of Br-BTN in the powder state can be explained by a very short fluorescence lifetime ( 2(I), 236 parameters, 1 restraints, 1.512 <  < 28.436 °, final R factors R1 = 0.0390 and wR2 = 0.0805, GOF = 1.048. CCDC deposit number: 1814396.

Crystallographic Data for Single Crystal Pyr-BTN. C21H18N2O1S1 Mr = 346.4, crystal dimensions 0.08 × 0.07 × 0.06 mm3, triclinic, space group P-1, a = 12.1809(9), b = 13.3983(9) Å, c = 16.9252(11) Å, = 72.327(3)°  = 71.229(4)° = 73.535(4)°, V = 2438.5(3) Å3, Z = 6, calcd = 1.415 g cm–3,  = 0.211 mm−1,  = 0.71073 Å (Mo K), T = 100(2) K, 7296 unique reflections out of 10052 with I > 2(I), 679 parameters, 0 restraints, 1.307 <  < 26.472 °, final R factors R1 = 0.0521 and wR2 =0.1176, GOF = 1.045. CCDC deposit number: 1814397.

4.4. Two-Photon Action Cross Section. TPACS values were measured following known method. Two equations are referred from the references as below. 𝑭(𝒕) cal 𝑭(𝒕) new

=

𝚽cal 𝜼2cal 𝝈2cal 𝑪cal 𝑷cal (𝒕) 𝟐 𝒏cal 𝚽new 𝜼2new 𝝈2new 𝑪new 𝑷new (𝒕) 𝟐 𝒏new

……(1)

The equation (1) is the main equation that calculates TPACS using a reference dye and equation (2) could be extracted from equation (1).

𝝈2new 𝝀 𝜼2new =

𝚽cal 𝜼𝟐cal 𝝈𝟐cal (𝝀)𝑪cal 𝑷cal (𝒕) 𝟐 𝑭(𝒕) new 𝒏cal 𝚽new 𝑪new 𝑷new (𝒕) 𝟐 𝑭(𝒕) cal 𝒏new

…….(2)

(σ2 = Two Photon Absorption Cross Section; η = Quantum Efficiency; σTPE (Two Photon Action Cross Section = ση; = Time Averaged Fluorescecne Emission; C =Fluorophore Concentration; = Time Averaged Laser Power; n=Reflective Index of Sample; Φ = Fluorescence Collection Efficiency) Φcal and Φnew are the identical value in the same experimental setup, and , are also identical when same laser has been applied. TPACS values of samples could be calculated by adding values of known TPACS (Two-Photon Action Cross Section) (ση), concentration (C), detected emission (), and known reflective index (n).



Page 20 of 26

Rhodamine B in methanol (100 μM) was used as a reference, and 100 μM of Pyr-BTN-OMe and Pyr-BTN in DMSO (dimethyl sulfoxide) were used for the measurement. Each refractive index of a given solvent was applied (assuming that the refractive index of sample is almost same as that of pure solvent). 100 μL of samples were loaded in well slides and covered with cover glass. The edge of cover glass was coated with transparent manicure to prevent the evaporation of solvent and then mounted on a vibration isolation table. Two-photon excitation was performed with a Tisapphire laser (Chameleon Vision II, Coherent) at 140 fs pulse width and 80 MHz pulse repetition rate. The emission intensity was collected through an HCX APO 10x objective lens (Leica, Germany) of a two-photon microscopy (TCS SP5 II, Leica, Germany) equipped with HyD detector (Leica, Germany), in the range of 400–665 nm.

4.5. Cytotoxicity Study. HeLa cells were thus seeded into 96-well plates at a density of 5000 cells/well and incubated for 24 h at 37 °C in a humidified atmosphere of 5% CO2 in air. The fluorophore solution with various concentrations (5, 10, 50 and 100 μM) was added into the culture media in the plate. The plate was incubated for 4, 8, 12 and 24 h and CCK-8 solution was added to each well of the plate. After the further incubation for 2 h, absorbance at 450 nm was measured using a microplate reader (Multiskan EX, Thermo Eletron). Cell viability was calculated by the ratio of the absorbance of control cells with that of dye-treated cells.

4.6. Cell Culture. HeLa human cervical cancer cells were obtained from Korean Cell Line Bank. HeLa cells were maintained in 10% (v/v) fetal bovine serum (FBS) and 1% (w/v) penicillinstreptomycin (PS) supplemented DMEM at 37 °C in a humidified atmosphere of 5% CO2 in air. Upon reaching approximately 80% confluence, cells were passaged. 4.7. Fluorescence Microscopic Imaging. Cells were seeded onto cell culture dish at a density of 1x105 cells. The cultured cells were incubated in DMEM-FBS buffer containing 50 μM of PyrBTN-OMe and Pyr-BTN NPs for 30 min at 37 °C under 5% CO2. Next, cells were washed with PBS to remove the remaining NPs, and then fixed with 4% formaldehyde solution. The images of the cells were recorded by confocal microscopy. Confocal fluorescence imaging experiments were performed on Leica TCS SP5 II Advanced System. The microscope was equipped with multiple laser lines (405, 458, 476, 488, 496, 514, 561, 594, 633 nm). 405 nm (for Pyr-BTN-OMe NPs) and 450 nm (for Pyr-BTN NPs) lasers were used for the confocal imaging experiments.


Page 21 of 26 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


Fluorescence signals were obtained through Hyd PMT (Hybrid detector Photo Multiplier Tube; Leica) in green channel (420−555 nm) for Pyr-BTN-OMe NPs and in yellow channel (555−620 nm) for Pyr-BTN NPs. Prepared cells were mounted on the tight-fitting holder. The imaging fieldof-view (FOV) was 9797 μm consisting of 10241024 pixels. Acquired images were processed by using LAS AF Lite (Leica, Germany).

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Methods and experimental details, optical properties of all the dyes in different organic solvents from nonpolar 1,4-dioxane to polar DMSO and phosphate buffer solution (PBS buffer pH 7.4), HOMO−LUMO energy calculation, crystalline state fluorescence for Pyr-BTN and Pyr-BTNOMe, side view crystal packing, TPACS graph, cytotoxicity value, copies of 1H &

13

C NMR,

crystallographic information file for C22H20N2O1S1 (CIF) and C21H18N2O1S1 (CIF). AUTHOR INFORMATION Corresponding Authors *Email: [email protected] *Email: [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENT This work was funded by the Global Research Laboratory Program (2014K1A1A2064569) through the National Research Foundation (NRF) funded by Ministry of Science, ICT & Future Planning, Republic of Korea. We thank Dr. Swapan Pramanik for determining the nanoparticles size with DLS.

REFERENCES 1.

Rajadurai, M. S.; Shifrina, Z. B.; Kuchkina, N. V.; Rusanov, A. L.; Muellen, K. Rigid Aromatic Dendrimers. Russ. Chem. Rev. 2007, 76, 767–783.



2.

Page 22 of 26

An, B. K.; Kwon, S. K.; Jung, S. D.; Park, S. Y. Enhanced Emission and Its Switching in Fluorescent Organic Nanoparticles. J. Am. Chem. Soc. 2002, 124, 14410–14415.

3.

Shifrina, Z. B.; Rajadurai, M. S.; Firsova, N. V.; Bronstein, L. M.; Huang, X.; Rusanov, A. L.; Muellen, K. Poly(Phenylene-pyridyl) Dendrimers: Synthesis and Templating of Metal Nanoparticles. Macromolecules 2005, 38, 9920–9932.

4.

Sreejith, S.; Carol, P.; Chithra, P.; Ajayaghosh, A. Squaraine Dyes: A Mine of Molecular Materials. J. Mater. Chem. 2008, 18, 264–274.

5.

Jana, A.; Sanjana, K.; Devi, P.; Maiti, T. K.; Singh, N. D. P. Perylene-3-ylmethanol: Fluorescent Organic Nanoparticles as A Single-Component Photoresponsive Nanocarrier with Real-Time Monitoring of Anticancer Drug Release. J. Am. Chem. Soc. 2012, 134, 7656–7659.

6.

Chandrasekhar, N.; Chandrasekar, R. Reversibly Shape-Shifting Organic Optical Waveguides: Formation of Organic Nanorings, Nanotubes, and Nanosheets. Angew. Chem. 2012, 124, 3616 –3621.

7.

Chandrasekhar, N.; Chandrasekar, R. Engineering Self-Assembled Fluorescent Organic Nanotapes and Submicrotubes from π-Conjugated Molecules. Chem. Commun. 2010, 46, 2915–2917.

8.

Basak, S.; Chandrasekar, R. Multiluminescent Hybrid Organic/Inorganic Nanotubular Structures: One-Pot Fabrication of Tricolor (Blue–Red–Purple) Luminescent Parallepipedic Organic Superstructure Grafted with Europium Complexes. Adv. Funct. Mater. 2011, 21, 667–673.

9.

Kim, H. J.; Lee, J.; Kim, T. H.; Lee, T. S.; Kim, J. Highly Emissive Self-assembled Organic Nanoparticles Having Dual Color Capacity for Targeted Immunofluorescence Labeling. Adv. Mater. 2008, 20, 1117–1121.

10. Kaeser, A.; Schenning, A. P. H. J. Fluorescent Nanoparticles Based on Self-Assembled π-Conjugated Systems. Adv. Mater. 2010, 22, 2985–2997. 11. Kumar, M.; George, S. J. Green Fluorescent Organic Nanoparticles by Self-Assembly Induced Enhanced Emission of a Naphthalene Diimide Bolaamphiphile. Nanoscale 2011, 3, 2130–2133. 12. Feng, L.; Zhu, C.; Yuan, H.; Liu, L.; Lv, F.; Wang, S. Conjugated Polymer Nanoparticles: Preparation, Properties, Functionalization and Biological Applications. Chem. Soc. Rev. 2013, 42, 6620−6633. 13. Zhang, X.; Li, S.; Liu, Z.; Wang, S.; Xiao, J. Self-Assembled Multicolor Nanoparticles Based on Functionalized Twistacene Dendrimer for Cell Fluorescent Imaging. NPG Asia Materials. 2015, 7, e230. 14. McVey, B. F. P.; Tilley, R. D. Solution Synthesis, Optical Properties, and Bioimaging Applications of Silicon Nanocrystals. Acc. Chem. Res. 2014, 47, 3045–3051. 15. Xu, S.; Li, D.; Wu, P. One-Pot, Facile, and Versatile Synthesis of Monolayer MoS2/WS2 Quantum Dots as Bioimaging Probes and Efficient Electrocatalysts for Hydrogen Evolution Reaction. Adv. Funct. Mater. 2015, 25, 1127–1136. 16. Chan, W. C.; Nie, S. Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic Detection. Science 1998, 281, 2016–2018. 17. Feng, L.; Zhu, C.; Yuan, H.; Liu, L.; Lv, F.; Wang, S. Conjugated Polymer Nanoparticles: Preparation, Properties, Functionalization and Biological Applications. Chem. Soc. Rev. 2013, 42, 6620–6633.


Page 23 of 26 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


18. Ding, D.; Li, K.; Liu, B.; Tang, B. Z. Bioprobes Based on AIE Fluorogens. Acc. Chem. Res. 2013, 46, 2441– 2453. 19. Genin, E.; Gao, Z.; Varela, J. A.; Daniel, J.; Bsaibess, T.; Gosse, I.; Groc, L.; Cognet, L.; Blanchard-Desce, M. “Hyper-bright” Near-Infrared Emitting Fluorescent Organic Nanoparticles for Single Particle Tracking. Adv. Mater. 2014, 26, 2258–2261. 20. Gao, Y.; Feng, G.; Jiang, T.; Goh, C.; Ng, L.; Liu, B.; Li, B.; Yang, L.; Hua, J.; Tian, H. Biocompatible Nanoparticles Based on Diketo-Pyrrolo-Pyrrole (DPP) with Aggregation-Induced Red/NIR Emission for In Vivo Two-Photon Fluorescence Imaging. Adv. Funct. Mater. 2015, 25, 2857–2866. 21. Shao, A.; Xie, Y.; Zhu, S.; Guo, Z.; Zhu, S.; Guo, J.; Shi, P.; James, T. D.; Tian, H.; Zhu, W. H. Far-Red and Near-IR AIE-Active Fluorescent Organic Nanoprobes with Enhanced Tumor-Targeting Efficacy: ShapeSpecific Effects. Angew. Chem. 2015, 127, 7383–7388. 22. Li, M.; Feng, L.; Lu, H.; Wang, S.; Chen, C. Tetrahydro[5]helicene-Based Nanoparticles for StructureDependent Cell Fluorescent Imaging. Adv. Funct. Mater. 2014, 24, 4405–4412. 23. Petkau, K.; Kaeser, A.; Fischer, I.; Brunsveld, L.; Schenning, A. P. H. J. Pre- and Postfunctionalized SelfAssembled π-Conjugated Fluorescent Organic Nanoparticles for Dual Targeting. J. Am. Chem. Soc. 2011, 133, 17063–17071. 24. Jana, A.; Nguyen, K. T.; Li, X.; Zhu, P.; Tan, N. S.; Agren, H.; Zhao, Y. Perylene-Derived Single-Component Organic Nanoparticles with Tunable Emission: Efficient Anticancer Drug Carriers with Real-Time Monitoring of Drug Release. ACS Nano 2014, 8, 5939–5952. 25. Mei, J.; Hong, Y.; Lam, J. W. Y.; Qin, A.; Tang, Y.; Tang, B. Z. Aggregation-Induced Emission: The Whole Is More Brilliant than the Parts. Adv. Mater. 2014, 26, 5429–5479. 26. Xue, X.; Zhao, Y.; Dai, L.; Zhang, X.; Hao, X.; Zhang, C.; Huo, S.; Liu, J.; Liu, C.; Kumar, A.; Chen, W. Q.; Zou, G.; Liang, X. J. Spatiotemporal Drug Release Visualized through a Drug Delivery System with Tunable Aggregation-Induced Emission. Adv. Mater. 2014, 26, 712–717. 27. Hu, Q.; Gao, M.; Feng, G.; Liu, B. Mitochondria-Targeted Cancer Therapy Using a Light-Up Probe with Aggregation-Induced-Emission Characteristics. Angew. Chem., Int. Ed. 2014, 53, 14225–14229. 28. Wang, Z.; Yong, T. Y.; Wan, J.; Li, Z. H.; Zhao, H.; Zhao, Y.; Gan, L.; Yang, X. L.; Xu, H. B.; Zhang, C. Highly Stable Near-Infrared Fluorescent Organic Nanoparticles with a Large Stokes Shift for Noninvasive Long-Term Cellular Imaging. ACS Appl. Mater. Interfaces 2015, 7, 3420–3425. 29. Zhang, C.; Wang, Z.; Tan, L.; Zhai, T. L.; Wang, S.; Tan, B.; Zheng, Y. S.; Yang, X. L.; Xu, H. B. A Porous Tricyclooxacalixarene Cage Based on Tetraphenylethylene. Angew. Chem., Int. Ed. 2015, 54, 9244–9248. 30. Moon, H.; Xuan, Q. P.; Kim, D.; Kim, Y.; Park, J. W.; Lee, C. H.; Kim, H. J.; Kawamata, A.; Park, S. Y.; Ahn, K. H. Molecular-Shape-Dependent Luminescent Behavior of Dye Aggregates: Bent versus Linear Benzocoumarins. Cryst. Growth Des. 2014, 14, 6613−6619. 31. Kwon, J. E.; Park, S. Y. Advanced Organic Optoelectronic Materials: Harnessing Excited-State Intramolecular Proton Transfer (ESIPT) Process. Adv. Mater. 2011, 23, 3615.



Page 24 of 26

32. Khan, A. U.; Kasha, M. Mechanism of Four-Level Laser Action in Solution Excimer and Excited-State Proton-Transfer Cases. Proc. Natl. Acad. Sci. U. S. A. 1983, 80, 1767. 33. Sakai, K.; Tsuzuki, T.; Itoh, Y.; Ichikawa, M.; Taniguchi, Y. Using Proton-Transfer Laser Dyes for Organic Laser Diodes. Appl. Phys. Lett. 2005, 86, 081103. 34. Zhao, J.; Ji, S.; Chen, Y.; Guo, H.; Yang, P. Excited State Intramolecular Proton Transfer (ESIPT): From Principal Photophysics to the Development of New Chromophores and Applications in Fluorescent Molecular Probes and Luminescent Materials. Phys. Chem. Chem. Phys. 2012, 14, 8803. 35. Wu, J.; Liu, W.; Ge, J.; Zhang, H.; Wang, P. New Sensing Mechanisms for Design of Fluorescent Chemosensors Emerging in Recent Years. Chem. Soc. Rev. 2011, 40, 3483. 36. Shynkar, V. V.; Klymchenko, A. S.; Kunzelmann, C.; Duportail, G.; Muller, C. D.; Demchenko, A. P.; Freyssinet, J.; Mely, Y. Fluorescent Biomembrane Probe for Ratiometric Detection of Apoptosis. J. Am. Chem. Soc. 2007, 129, 2187. 37. Park, S.; Kwon, J. E.; Kim, S. H.; Seo, J.; Chung, K.; Park, S.; Jang, D.; Medina, B. M.; Gierschner, J.; Park, S. Y. A White-Light-Emitting Molecule: Frustrated Energy Transfer Between Constituent Emitting Centers. J. Am. Chem. Soc. 2009, 131, 14043. 38. Peng, L.; Xu, S.; Zheng, X.; Cheng, X.; Zhang, R.; Liu, J.; Liu, B.; Tong, A. Rational Design of a RedEmissive Fluorophore with AIE and ESIPT Characteristics and Its Application in Light-Up Sensing Of Esterase. Anal. Chem. 2017, 89, 3162−3168. 39. Yao, H.; Funada, T. Mechanically Inducible Fluorescence Colour Switching in the Formation of Organic Nanoparticles of an ESIPT Molecule. Chem. Commun. 2014, 50, 2748–2750. 40. Xiao, H.; Chen, K.; Cui, D.; Jiang, N.; Yin, G.; Wanga, J.; Wang, R. Two Novel Aggregation-Induced Emission Active Coumarin-Based Schiff Bases and Their Applications in Cell Imaging. New J. Chem. 2014, 38, 2386–2393. 41. Kundu, A.; Hariharan, P. S.; Prabakaran, K.; Moon, D.; Anthony, S. P. Aggregation Induced Emission of Excited-State Intramolecular Proton Transfer Compounds: Nanofabrication Mediated White Light Emitting Nanoparticles. Cryst. Growth Des. 2016, 16, 3400−3408. 42. Kim, Y. H.; Roh, S. G.; Jung, S. D.; Chung, M. A.; Kim, H. K.; Cho, D. W. Excited-State Intramolecular Proton Transfer on 2-(2'-Hydroxy-4'-R-Phenyl)Benzothiazole Nanoparticles and Fluorescence Wavelength Depending on Substituent and Temperature. Photochem. Photobiol. Sci. 2010, 9, 722–729. 43. Roy, B.; Rao, A. S.; Ahn, K. H. Mononuclear Zn(II)- and Cu(II)-Complexes of a HydroxynaphthaleneDerived Dipicolylamine: Fluorescent Sensing Behaviours Toward Pyrophosphate Ions. Org. Biomol. Chem. 2011, 9, 7774–7779. 44. Ma, D.; Cai, Q. Copper/Amino Acid Catalyzed Cross-Couplings of Aryl and Vinyl Halides with Nucleophiles. Acc. Chem. Res. 2008, 41, 1450–1460. 45. Wang, R.; Liu, Di.; Xu, K.; Li, J. Substituent and Solvent Effects on Excited State Intramolecular Proton Transfer in Novel 2-(2′Hydroxyphenyl)Benzothiazole Derivatives. J. Photochem. Photobiol. A: Chemistry 2009, 205, 61–69.


Page 25 of 26 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


46. Ghiggino, K. P.; Scully, A. D.; Leaver, I. H. Effect of Solvent on Excited-State Intramolecular Proton Transfer in Benzotriazole Photostabilizers. J. Phys. Chem. 1986, 90, 5089-5093. 47. Chang, S. M.; Hsueh, K. L.; Huang, B. K.; Wu, J. H.; Liao, C. C.; Lin, K. C. Solvent Effect of Excited State Intramolecular Proton Transfer in 2-(2′-Hydroxyphenyl) Benzothiazole upon Luminescent Properties. Surf. Coat. Technol. 2006, 200, 3278–3282. 48. Kwak, M. J.; Kim, Y. Photostable BF2-Chelated Fluorophores Based on 2-(2′-Hydroxyphenyl)benzoxazole and 2-(2′-Hydroxy-phenyl)benzothiazole. Bull. Korean Chem. Soc. 2009, 30, 2865–2866.



Page 26 of 26

TOC

-Me S

ESIPT

N

S

N

OMe

OH

R

R BTN-OMe

BTN

R = CN

CN-BTN

Br-BTN -OMe

R = Br

Br-BTN

R=

N

NH O

R

CN-BTN -OMe

Pyr-BTN -OMe

S

Pyr-BTN


Exploration of Molecular Shape-Dependent Luminescence Behavior

Recommend Documents