Synthesis, characterization and photophysical properties of meso-indole-boron-dipyrromethene derivatives and their cell imaging and viscosity sensing

Dyes and Pigments 164 (2019) 156–164

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Synthesis, characterization and photophysical properties of meso-indoleboron-dipyrromethene derivatives and their cell imaging and viscosity sensing

T

Ben Dong, Kailiang Zhong, Yun Lu∗ Department of Polymer Science and Engineering, State Key Laboratory of Coordination Chemistry, Collaborative Innovation Center of Chemistry for Life Sciences, Key Laboratory of High Performance Polymer Materials and Technology (Nanjing University), Ministry of Education, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210093, PR China

A R T I C LE I N FO

A B S T R A C T

Keywords: meso-indole boron-dipyrromethene derivatives Fluorescence characteristics Photoluminescence Cell imaging Viscosity sensing

Based on the molecular design and structural modification of boron-dipyrromethene (BODIPY), two new BODIPY derivatives with meso-substituted functional indole moieties were successfully prepared. These new compounds were characterized by NMR, HRMS and FTIR, exhibiting the different UV–vis and PL spectral phenomena because of their varied molecular structures in various solvents and different degrees of negative solvatochromism. Theoretical calculations and CV results show that the substituted NO2 group at 5-position of indole is favorable for increasing the oxidation potential of the molecules, while the NH2 group is the opposite. These two BODIPY derivatives have different sensitivities to pH changes, and can be appropriately applied as cell imaging materials due to their good physical chemical characteristics and excellent biocompatibility. In particular, the fluorescence characteristics of BODIPY derivative with the substituted NH2 group at 5-position of indole show a good solvent viscosity dependence, highlighting its potential application for testing the intracellular viscosity changes of the living cell in disease diagnosis.

1. Introduction Cellular viscosity is recognized as a vital factor to numerous diffusion-mediated cellular processes, and its abnormal changes relate to many diseases [1–3], apoptosis or suffering from cancer [4]. To better know the pathological influences related to the abnormal viscosity levels in biological systems and exploit the therapeutic strategies with viscosity correlation, it is fairly essential to develop the efficient and reliable methods to detect viscosity changes in various biological systems. Indoles are a class of most important and widely distributed nitrogenous heterocyclic compounds because of their ubiquity in natural products, drugs, bioactive compounds and other functional molecules [5]. Numerous efforts have been actively pursued for the construction of various indole materials during the past several decades. For instance, the indole oligomer was placed in a luminance device as a lightemitting material, showing the effective electroluminescence quantum efficiency and the luminous efficiency [6]. The dye-sensitized solar cells based on the organic dyes containing substituted indole was constructed in a typical sandwich-type cell, presenting efficient photon-toelectron conversion properties [7]. Although much work about indole-

∗

based material was reported, it remains challenging to develop more meaningful materials with novel properties for satisfying the needs of many fields, especially the rarely studied viscosity sensing. On the other hand, boron-dipyrromethene (BODIPY) fluorophores are of particular interest due to their excellent photophysical properties including large extinction coefficients, high molar absorption coefficients, narrow emission bandwidths, as well as good stability and solubility in various physiological conditions. More importantly, the BODIPY core can be fine-tuned by introducing appropriate substituents at different position of the BODIPY backbone [8–12], and thus extensively used in chemo-sensors, biology cell-imaging, material science, environmental sciences and the other fields [13–15]. For example, a fluorescent probes based on 4,4-difluoro-8-(4-hydroxyphenyl)-1,5-dimethyl-4-bora-3a,4a-diaza-s-indacene was fabricated, and its PL intensity excited by visible light increased linearly within the pKa range of 7.5–9.3 [16]. The 4,4-difluoro-8-(4-methylphenyl)-3-azido-4-bora3a,4a-diaza-s-indacene was used as a fluorescence turn-on probe for detecting H2S rapidly [17]. Also a fluorescent probe based on the 4,4difluoro-8-(4-aminebiphenyl)-1,5-dimethyl-4-bora-3a,4a-diaza-s-indacene was made and applied to evaluate and identify cancer, normal

Corresponding author. E-mail address: [email protected] (Y. Lu).

https://doi.org/10.1016/j.dyepig.2019.01.017 Received 2 October 2018; Received in revised form 12 January 2019; Accepted 13 January 2019 Available online 14 January 2019 0143-7208/ © 2019 Elsevier Ltd. All rights reserved.

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2.3. Electrochemical testing

and apoptotic cells according to the changes of intracellular viscosity [1]. These previous studies indicate that the intramolecular charge transfer occurred in BODIPY fluorophore and the functional groups substituted at meso-position may have important effect on the performance of the material. Also, the functional groups with strong electron donating group could tend to strong intramolecular charge transfer, quenching the fluorescence and resulting in the lower photoluminescence (PL) quantum yield [18–21]. It is thus expected to make reasonable molecular design for new BODIPY fluorophores with novel conjugated structures so as to deepen the understanding of the relationship between structure and property and thus further develop the valuable functional materials. In this work, two BODIPY derivatives containing both BODIPY fluorophores and the indole moieties with NO2 and NH2 groups, c1 and c2, were designed and synthesized successfully. Characterizations of samples include UV spectra, fluorescence emission spectra, stokes shift, molar extinction coefficient and PL quantum yield. We also investigated the electronic properties of these BODIPYs involving the oxidation potential, PL intensity, HOMO-LUMO gaps (ΔECV) and photostability, the effect of NO2 and NH2 at 5-position of the indole moieties on the electrochemical and optical properties as well as the theoretical calculation of these resultant structures. In terms of the quite low toxicity, excellent biocompatibility, good PL quantum yield and high physical and chemical stability of these prepared BODIPYs, some potential applications in live cell imaging and viscosity detecting were explored.

Cyclic voltammetry experiment was carried out through threeelectrode setup including a platinum sheet as the working electrode, platinum wire as the counter electrode, Ag/AgCl as the reference electrode. Experiments were run at 50 mV s−1 scan rates in 0.1 M tetrabutylammonium hexafluorophosphate as the supporting electrolyte. The electrochemical energy gap (Eg in eV) of the samples were calculated according to the following equations [22]:

EHOMO = −e(Eonset − Eonset ox ferrocene + 4.8) (eV) ELUMO =

−e(Eonset red

−

Eonset ferrocene

+ 4.8) (eV)

Eg = ELUMO- EHOMO

(1) (2)

(3)

where the Eox and Ered were respectively the onset of oxidation and reduction potential. 2.4. Cellular toxicity and cellular imaging tests The testing process of biocompatibility and biomedical imaging of the samples were provided in SI in details. 2.5. Photostability The photostability was measured in l cm width quartz cells where the sample solution was irradiated under a 500 W I–W lamp at room temperature [23]. A cold trap (8 L solution of 60 g/L NaNO2 and 25 cm in length) was set up between the lamp and the cells to avoid heat and absorbance of short wavelength light. The distance between the samples and the lamp was 30 cm. The irreversible bleaching of samples at absorption peak was monitored as a function of time. All samples (1.0 × 10−5 mol/L in PBS buffer solution) were deoxygenated with N2 for 10 min.

2. Materials and methods 2.1. Materials Unless otherwise noted, materials obtained from commercial suppliers were used without further purification. 5-Nitroindole, 2,4-dimethyl-1H-pyrrole, 2,3-dicyano-5,6-dichlorobenzoquinone (DDQ) and Et3N were procured from Meryer Chemical Technology Co., Ltd. (Shanghai, China), 1-bromohexane and indole were purchased from Aladdin Chemical Co., China, and boron trifluoride diethyl etherate was supplied from Nanjing Chemical Reagent Co., China. All analytical grade solvents were purchased from commercial suppliers and stored anhydrously over activated molecular sieves or freshly distilled under nitrogen from an appropriate drying agent.

2.6. Theoretical calculations Theoretical calculations on the geometrical and electronic properties were performed on the Gaussian 03 program package by using the density functional theory (DFT) method at B3LYP/6-31G (d) level. Molecular orbitals were visualized using GaussView.

2.2. Characterizations 2.7. pH stability test Fourier transform infrared spectra (FTIR) were collected on a Bruker VECTOR22 spectrometer. 1H and 13C NMR spectra were recorded on a Bruker AVANCE III 400 MHz spectrometer. Chemical shifts (δ) are provided in ppm relative to CDCl3 (7.26 and 77.23 ppm for 1H and 13C respectively), DMSO-d6 (2.50 ppm and 40.0 ppm for 1H and 13C respectively) or to internal TMS. The UV–vis and emission spectra were recorded on Perkin Elmer Lambda 35 instrument and FluoroMax Spectrofluorometer (HORIBA JobinYvon, France) respectively. Highresolution mass spectra (HRMS) were given by using the Agilent 6540 high-resolution mass spectrometer in positive mode. The absolute luminescence quantum yield (ΦF) was measured by a FLS920 fluorescence spectrophotometer. The single-crystal X-ray diffraction data for c1 was recorded on a BRUKER D8 VENTURE system with Cu-Kα radiation (λ = 1.54178 Å). Differential Fourier synthesis method is used to solve the molecular structure directly. All of the non-hydrogen atoms (C, N, O, B, F) are anisotropically refined by the least square method on F2 using the SHELXTL package. The structure picture was made of diamond 3.2. CCDC1867173 contained additional crystallographic data for this work. The quantum yield (Φ) of the BODIPYs were measured by using the fluorescein (in 0.1 M NaOH, literature quantum yield 85%) as standard and calculated using the formula of Φ = ΦR × (I/IR) × (AR/ A) × (n2/nR2) [22].

The pH titration experiments were carried out in 0.1 M citrate buffer solution, in which the pH of the buffer solution was adjusted to basic or acidic with 0.1M NaOH or 0.1 M HCl respectively, and was monitored with a digital pH meter equipped with a glass biotrode electrode. Calibration of the instruments was performed with standard aqueous solutions of pH 4.00 and 9.18 in the potassium hydrogen phthalate and boric acid buffer solution respectively. The solution of the c1 or c2 in 0.1 mL dioxane was mixed with 0.1 M citrate buffer with an appropriate pH. The final concentration of c1 or c2 in the test solution was 10−5 M. 2.8. Syntheses of BODIPYs with the meso-indole substituted by NO2 or NH2 group The overall approaches to the synthesis of BODIPYs with the mesoindole substituted by NO2 or NH2 group were depicted in Scheme 1, and the obtained two compounds were referred to as BODIPYs for short, and named as c1 and c2 respectively. The general procedure for the preparation of c1 and c2 was presented as follows. The related compounds were denoted as a1 and b1 and synthesized according to the previous related literature [6,24,25], (see the SI materials for details). c1: Indole-3-carboxaldehyde (0.55 g, 2.0 mmol) was added to a 157

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Scheme 1. Synthetic routes for c1 and c2.

3. Results and discussion

solution of 2, 4-dimethylpyrrole (0.38 g, 4.0 mmol) in dry dichloromethane (100 mL) in the dark under a nitrogen atmosphere (N2). Then 30 mg trifluoroacetic acid (TFA) was added into the solution under N2. After stirring the mixture at room temperature for 24 h, DDQ (0.46 g, 2.0 mmol) was added and stirred for another 1 h under 0 °C. Then triethylamine (TEA) (8 mL) and BF3·Et2O (8 mL) were added slowly to the solution. After that, the mixture was stirred for 6 h at room temperature, then quenched with water, and stirred additional 30 min. The aqueous was extracted with chloroform (3 × 50 mL). The combined organic layer was washed with brine and dried over Na2SO4. After completely removing the solvent, the residue was purified by silica gel chromatography with petroleum/ethyl acetate (10:1 v/v) as eluent to give red orange solid, and the obtained compound was recrystallized from CH2Cl2/hexane to afford the desired compound as an orange-red needled solid in 25.2% yield (248 mg). 1H NMR (400 MHz, CDCl3) δ 8.38 (d, 1H, J = 2.10 Hz), 8.17 (dd, 1H, J = 2.20 Hz, J = 9.10 Hz), 7.47 (d, 1H, J = 9.20 Hz), 7.19 (s, 1H), 5.98 (s, 2H), 4.26 (t, 2H, J = 7.04 Hz), 2.57 (s, 6H), 1.93–1.85 (m, 2H), 1.34 (s, 6H), 1.33–1.26 (m, 6H), 0.86 (t, 3H, J = 7.00 Hz). 13C NMR (100 MHz, CDCl3) δ 156.0, 142.7, 139.0, 132.9, 132.8, 129.5, 126.7, 121.6, 118.4, 117.0, 112.0, 110.2, 47.4, 31.5, 30.8, 26.7, 22.7, 14.8, 14.5, 14.1. FTIR (KBr, cm−1): 3108, 2927, 2856, 1622, 1546, 1512, 1470, 1334, 1194, 1153, 978. MS (ESI) Calcd for C27H31BF2N4O2 [M+Na]+: 515.24; Found: 515.50. HRMS (ESI) Calcd for C27H31BF2N4O2 [M+H]+: 493.2581; Found: 493.2589. c2: a mixture of c1 (0.25 g, 0.5 mmol), iron powder (0.39 g, 7.0 mmol) and chloric acid methanol solution (2.5 mL, 0.6 mol/L) in mixed solution of methanol (10 mL) and H2O (5 mL) was heated to reflux for 2 h. The reaction mixture was cooled and filtered after the reaction was confirmed to be complete by TLC monitoring. The filtrate was evaporated and the crude product was purified on silica gel chromatography with petroleum/ethyl acetate (10:1 v/v) as eluent to give red solid (247 mg), and the obtained compound was recrystallized from CH2Cl2/hexane to afford a red needled solid in 97% yield (223 mg). 1H NMR (400 MHz, DMSO-d6) δ 7.30–7.21 (m, 2H), 6.58 (dd, 1H, J = 2.00 Hz, J = 8.70 Hz), 6.39 (d, 1H, J = 1.90 Hz), 6.12 (s, 2H), 4.64 (brs, 2H), 4.14 (t, 2H, J = 6.80 Hz), 2.45 (s, 6H), 1.78–1.69 (m, 2H), 1.44 (s, 6H), 1.28–1.18 (m, 6H), 0.81 (t, 3H, J = 6.80 Hz). 13C NMR (100 MHz, DMSO-d6) δ 154.0, 143.3, 142.9, 137.9, 132.6, 130.3, 127.9, 127.4, 121.2, 113.0, 111.1, 106.0, 101.4, 46.1, 31.3, 30.7, 26.2, 22.5, 14.7, 14.5, 14.4, 14.3. FTIR (KBr, cm−1): 3471, 3376, 3098, 2930, 2856, 1630, 1546, 1468, 1306, 1196, 1069, 981. MS (ESI) Calcd for C27H34BF2N4 [M+H]+: 463.28; Found: 463.58. HRMS (ESI) Calcd for C27H34BF2N4 [M+H]+: 463.2839; Found: 463.2838.

3.1. Synthesis BODIPY derivatives have been received various investigation because of their unique optical and chemical properties which could be fine-tuned by the modification of chemical structures with various substituents. In our case, BODIPY is chose as core backbone in which indole molecule was introduced at the meso-position to improve the electronic and photophysical properties of the conjugated compounds. Also, the NO2 and NH2 groups were linked respectively into 5-position of the indole moiety to regulate the electronic push-pull ability of whole molecule. And at the same time, the hexyl was used to substitute the H of NH in the indole in order to get good solubility. In this work, two BODIPYs c1 and c2 containing both BODIPY fluorophores and indole moieties with NO2 or NH2 groups were synthesized (Scheme 1). The molecular structure and purity of the products were confirmed by the NMR, HRMS and IR spectroscopies. In common organic solvents, the as-prepared compounds c1 and c2 showed good solubility.

3.2. X-ray crystal structure of c1 Single crystals of BODIPYs c1 and c2 were cultivated from the slow evaporation of their dichloromethane/hexane solutions at room temperature and atmospheric pressure, but only c1 offered its single crystal. Fig. 1 illustrates the X-ray crystal structure of c1 with a triclinic form and a space group of P1. The data refinement parameters shown in Tables S1–3 (Supporting Information) confirm that BODIPYs c1 possess a slightly distorted tetrahedral geometry around boron atom, and the values of N1-B-N2 and F1-B-F2 angles are 108.6° and 117.0° respectively, indicating a normal delocalization of positive charge [26]. The dihedral angle between the indole rings and BODIPY core is 95.4°, slightly higher than that of the already reported meso-phenyl BODIP [27]. This is a result of the introduction of the NO2 group to the 5position of the indole, increasing the free rotation degree of the indole and BODIPY core [23]. Fig. 2 shows head-to-tail antiparallel π-π stacking between BODIPY cores separated at 3.90 Å in the crystals of c1, few intermolecular π-π stacking is observed because of the antiparallel arrangement between NO2 group and indole ring at a distance of 3.70 Å [25]. BODIPYs c1 forms the multiple C−H···F intermolecular hydrogen bonds owing to the interaction of F atoms with strong electron negativity and H atoms provided by BODIPY core [28–30], triggering the establishment and formation of various crystal packing structure or the interesting hydrogen bonding networks [27]. 158

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The emission spectra of c1 and c2 in different solvents were investigated at room temperature [33]. Their normalized emission spectra are illustrated in Fig. 4. In the case of c1, a dual peak or a peak with shoulder in 1,4-dioxane, THF, CHCl3, EtOH and DMF is observed. Among these dual peak, the peak at about 502 nm should be attributed to locally excited (LE) state, while that at 550∼579 nm could be assigned to the twisted intermolecular charge transfer (TICT) state [34]. The different redshift phenomena of emission are observed in these solvents, indicating the presence of negative solvatochromism (Fig. 4a) [35]. In the solvents of CH3CN and hexane, c1 shows a single emission peak at around 517 nm. The varied PL spectra for c1 in different polarity solvents probably relate to the energy surfaces of the different surroundings (Fig. 5) as well as the electronic effect. In polar solvents, the emission of c1 comes primarily from the TICT state that possesses the highest energy. When the solvent polarity decreases, the TICT and LE states with lower energy are stabilized via solvation causing these two levels to move away from one another [34]. Similar spectral changes are observed for c2 in polar solvents (CH3CN, DMF and EtOH) and mid-polar solvents (1,4-dioxane, THF, CHCl3). In contrast, the PL spectra of c2 exhibit a larger red shift for its emission peak to 576 nm in hexane (Fig. 4b). These results indicate that the introduction of NH2 groups at 5-position of the indole acted electronic donor, and the more stronger electron-donating effect makes the intramolecular charge transfer in mid-polar and polar solvents much larger than that in nonpolar solvent [15]. The solid-state fluorescence emission peaks of c1 and c2 are located at 550 and 590 nm (Fig. 4c), and the PL quantum yield are 17.8% and 0.9%, respectively. Such results suggest that luminous behaviors of asprepared AIE molecules containing the indole-BODIPY bichromophores could be fine-tuned by changing the substituents on indole, thus adjusting the intramolecular electronic effect [7]. Collating all the test results, the photophysical results of c1 and c2 are listed in Table 1. It can be seen that c1 shows the highest (62.2%) and lowest (11.6%) quantum yield in THF and CH3CN respectively, implying the existence of the equilibrium between the TICT and LE the state. While in THF and 1,4-dioxane, c1 is less in TICT state and chiefly in LE state which has a fast radiative rate constant, leading to the higher quantum yields. When polarity increases, the excited state changes into the TICT state, and emission is weakened due to the sensibility of the TICT state to all kinds of energy consumption processes, resulting in the lower quantum yield in the solvents of CH3CN and EtOH. Similar change in quantum yield could be noticed for c2 in polar solvents (CH3CN, DMF and EtOH) and mid-polar solvents (1,4-dioxane, THF, CHCl3). By contrast, c2 showed a highest quantum yield of 50.4% in hexane (Fig. 4b and Table 1). This can be ascribed to that c2 was mainly in LE state which inhibits the photoelectron transfer in molecules leading to the decrease in energy of intramolecular rotation, electron vibrational relaxation and internal conversion. The emission features effected by the solvents also can be evaluated through the direct correlations by the relationship between the solvent

Fig. 1. Molecular structure (ORTEP diagram) of c1.

It can be seen from the molecular structure information and DFT results (Fig. S1), c2 possesses a smaller dihedral angle of 81° than c1 (91°) between the meso-indole and the BODIPY core due to the smaller steric effects of NH2 group. The larger dihedral angles, for c1, can suppress the efficiency of internal conversion through intramolecular vibronic relaxation and freely rotation in solvents as well as the consumption of excitation energies, leading to the enhanced fluorescence quantum yields [31]. On the basis of c1, the three-dimensional network also can be speculated to form through a large number of intra/intermolecular hydrogen bonds in BODIPYs c2. 3.3. Solvatochromism photophysical properties The normalized absorption spectra of BODIPYs c1 and c2 in different solvents are depicted in Fig. 3. It can be seen that both absorption spectra of c1 and c2 show a broad absorption with three obvious bands. The first absorption at around 280–330 nm could be ascribed to localized π-π* transition of the electron-rich donors of indole moieties, the second at about 350–420 nm could be assigned to the S0→S2 (π-π*) transition of BODIPY core [29], and the third at around 450–550 nm could be attributed to a mixture of S0→S1 (π-π*) transition and the intramolecular charge transfer (ICT) transition between indole moieties with substituents and the BODIPY core [32]. Furthermore, the absorption spectra of c1 and c2 are not remarkably solvent-dependent, showing that the influence of solvent polarity is inappreciable for the compounds in the ground state.

Fig. 2. Packing structure of c1 single crystal, (a) distance of BODIPY core, (b) distance of indole. 159

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Fig. 3. UV–vis absorption spectra of (a) c1 and (b) c2 in various solvents.

Fig. 4. The normalized PL spectra of c1 (a) and c2 (b) in different solvents (1.0 × 10−5 M), (c) solid-state PL spectra of c1 and c2.

these BODIPYs obviously. For the electron-donating group of NH2, after irradiation for 4 h, the absorbance of c2 declines about 27% of its initial intensity. In contrast, for the electron-withdrawing group of NO2, the absorbance of c1 remains almost constant and only lost 7% of its initial intensity. The difference in photostability is probably because the BODIPYs have different electron cloud density due to the electronic effect of NO2 and NH2 which makes them have different photofading speed under the irradiation [23]. This observation clearly indicate that the photostability of these compounds could be effectively fine-tuned by the introduction of the different electron donating or withdrawing group at the 5-position of indole.

polarity parameter (Δf), the absorption stokes shift and emission maxima via Lippert–Mataga formulas [36].

Δv = Δvabs − Δvem =

2 2Δf (μ − μ g) + constant hca3 e

(4)

and

Δf = f (ε )− f (n2 )≈

ε−1 n2 − 1 − 2ε + 1 2n2 + 1

(5)

The resulted data including dielectric constants, refractive indices and Δf parameters for c1 and c2 were summarized in Table S3. By plotting Δυ vs Δf, as shown in Fig. 6, an obvious negative solvent effect for both c1 and c2 can be observed from the slops of the fitting straight lines. In other words, with the increase of solvent polarity, blue shifts of the emission peaks for c1 and c2 solution occur, most likely because in polar solvents, c1 or c2 is mainly presented in the TICT state and involved in all kinds of nonradiative energy consumed processes [36,37]. The above results further reveal that c1 and c2 possess polar nature and stronger TICT effect in the excited state, which are beneficial to nonlinear optical properties [38].

3.5. Electrochemical analysis Cyclic voltammetry (CV) was employed to understand the electrochemical properties and redox behaviors of the BODIPYs c1 and c2 and estimate their HOMO and LUMO energy levels (Table 2 and Fig. S2). It can be seen that the oxidation potentials of c1 is higher than that of c2. This may be ascribed to the different electron donating or withdrawing groups leading to the different π-π stacking interactions between neighboring indole and BODIPY core [23]. The HOMO energy levers were determined to be −5.48 eV and −5.39 eV for c1 and c2, respectively, corresponding to the difference charge transfer strengths of NO2 and NH2 at 5-substituent indoles. Moreover, the determined electrochemically HOMO-LUMO gaps (ΔECV) are 2.13 eV and 2.55 eV for c1 and c2, suggesting that the band gap could be adjusted effectively by the indole moieties with different 5-substituents. On the other hand, the smaller band gap of the c1 could be attributed to the influence of the strong electron-withdrawing group of NO2 leading to the reduced

3.4. Photostability Photostability of fluorescence compound is one of the most important characters for practical applications [28]. The photofading process of BODIPYs c1 and c2 were tracked by detecting the decrease of their maximal absorptions in PBS solutions at different times, and the results are summarized in Fig. 7. It can be seen that different substituents at the 5-position of indole moiety affect the photostability of 160

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Fig. 5. Schematic of the describing the LE→TICT state. Table 1 Photophysical data of BODIPYs c1 and c2 in different solvents with a concentration of 5 × 10−6 mol/L. BODIPYs

Solvent

λ(ab) nm

λ(em) nm

Δλ nm

ε 105 M−1cm−1

Φf (%)

c1

DMF CH3CN EtOH Dioxane CHCl3 THF Hexane DMF CH3CN EtOH Dioxane CHCl3 THF Hexane

506 504 506 507 510 507 508 502 500 502 503 506 502 504

540 517 521 562 542 555 523 521 517 520 527 527 522 576

34 13 15 55 32 48 15 19 17 18 24 21 20 72

0.22 0.17 0.24 0.23 0.25 0.23 0.25 0.94 0.93 0.96 1.05 0.99 1.00 1.05

44.0 11.6 21.3 59.8 45.3 62.2 16.3 0.2 0.5 3.3 11.9 2.9 7.0 50.4

c2

intermolecular π-π stacking between the antiparallel BODIPY units. Fig. 6. Lippert-Mataga solvent polarity graphs for c1 and c2.

3.6. Computational calculations behaviors of c1 and c2 in solution. Additionally, the theoretical HOMOLUMO gaps for c1 and c2 are 2.95 eV and 3.04 eV respectively, consistent with the CV experimental data (Table 2 and Fig. S2). Therefore, according to the experimental and theoretical results, the electronic structure and the optical properties of these indole-BODIPY compounds could be adjusted effectively through rational molecular design.

The HOMO and LUMO obtained via theorical calculation are shown in Fig. 8. In the case of c1, The HOMO electron density is found to be distributed on the BODIPY core and parts of methyl of BODIPY moiety, while the LUMO electron density is mainly distributed over the BODIPY unit and parts of the pyrrole of indole moiety. Such electronic distribution indicate the intramolecular charge transfer from the electronrich methyl moiety to the electron-deficient BODIP core [39]. In the contrast, the HOMO and LUMO electronic distribution for c2 are on the indole unit and the BODIPY core, and the HOMO and LUMO energy levels are separated well, demonstrating the strong charge migration between the indole and BODIPY core [25]. Such different HOMO and LUMO electron density distribution is in agreement with the emission

3.7. pH stability The influence of protonation and deprotonation on the emission behaviors of c1 and c2 with indole moiety was evaluated in different pH conditions. The fluorescence intensity of c1 corresponds obviously 161

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show that the c2 could be homogeneously dispersed in mediums with different pH values with an almost invariable average diameter size, for examples, 104.0 nm (pH = 1.43), 103.2 nm (pH = 2.54), 102.4 nm (pH = 3.52), 104.8 nm (pH = 4.55), 104.2 nm (pH = 5.57) and 103.6 nm (pH = 7.29). This fact further proves that the -NH2 group at 5-position of the indole unit is completely protonated under strong acidic condition, suppressing the intramolecular charge transfer and consequently resulting in the increase in fluorescence emission. Moreover, such an increase in fluorescence intensity is nearly linear, demonstrating the potential of c2 as pH sensor in the aqueous media with pH < 5. When the pH is > 5.0, the fluorescence intensity is at a fairly low level, suggesting that the c2 have good stability in basic conditions [41].

3.8. Live-cell imaging The organic conjugated fluorescent materials can be applied as biomedical imaging materials due to their excellent photoelectric properties, low cytotoxicity and good biocompatibility. Due to the good physical and chemical stability and high fluorescence quantum efficiency of c1 and c2, the biocompatibility were investigated in order to understand their application possibility in the living cells imaging. Studies on cytotoxicity and cell imaging reveal that for c1 and c2 the cell viability is above 89% and 90% even at a concentration of 10 μg mL−1 (Figs. S4a and S5a), suggesting their low toxicity to the HL60 cells. The c1 and c2 are found only in the cell membrane and cytoplasmic area of the cell but very weak at the central region corresponding to the nucleus, implying that these compounds could easily penetrate into the cell but not into the nuclei (Figs. S4c and S5c), thus avoiding effectively genetic disruption. Furthermore, no morphological cell damage is observed upon incubation with the samples, further exemplifying its low cytotoxicity. Therefore, c1 and c2 are well suitable for biological applications such as live-cell imaging, protein analysis, cell tracking, isolation of biomolecules etc.

Fig. 7. Photostability of BODIPYs c1 and c2 (1.0 × 10−5 M) in PBS represented by the change of absorption maximum wavelength with the irradiation time. Table 2 Electrochemical data of c1 and c2a. Entry

Eoxb (V)

Eredb (V)

HOMO(eV)

LUMO(eV)

Egc(V)

c1 c2

1.08 0.99

−1.05 −1.56

−5.48 −5.39

−3.35 −2.84

2.13 2.55

a E(Fc+/Fc) vs. Ag/AgCl with a potential of 0.44 V was used as the internal reference. b Onset oxidation or reduction potential vs. Ag/AgCl. c Band gaps.

3.9. Effect of viscosity The fluorescence spectrum of c2 in ethanol exhibit a weak emission at 519 nm when being excited at 489 nm. This is because free rotation of meso-substituted indole rings enhances the consumption of the energy and the photo-induced electron transfer from the lone pair of electrons on nitrogen of the indole to the photo-excited BODIPY moiety and results in the fluorescence quenching [42]. Interestingly, with the viscosity increasing from 3.42 to 769.00 cP (ethanol: glycerol, 0–90%) (Table S4), the fluorescence peak intensity of c2 at 517 nm increased 2.5 folds (Fig. 10). Furthermore, the Fröster-Hoffmann equation was utilized [43] to correlate the relationship between the emission intensity of c2 and the solvent viscosity. log I = C + x log η

(6)

where η, I, and x correspond to viscosity, emission intensity and sensitivity of the probe to viscosity respectively, and C is a constant. By plotting log I vs log η, a linear enhanced relationship (R2 = 0.9931, with a coefficient x of 0.18) is obtained with increasing solvent viscosity. These results can be attributed to the role of the amino group making the indole ring as an efficient molecular rotor by providing a reasonable and available D–π–A system in viscous medium. As rotation get restricted, the photo-induced electron transfer also become unfavorable, causing the increase in fluorescence emission [1]. The viscosity dependence of fluorescence intensity shown by c2 demonstrates that c2 can be appropriately applied to detect the viscosity changes in living cells due to its outstanding characteristics such as good biocompatibility and well linear relationship between log I and log η.

Fig. 8. DFT calculated HOMO, LUMO of c1 and c2 with corresponding energy gap.

with the varied pH value from 2 to 13 (Fig. 9a and b), which is mainly due to the protonation and deprotonation reaction taken place at the N position of indole or the BODIPY core under the different pH conditions [40]. In the case of c2, the fluorescence is enhanced strongly with decrease of pH from 4.55 to 1.42(Fig. 9). In addition, DLS results (Fig. S3) 162

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Fig. 9. (a)–(d) Changes in the PL intensity of c1 and c2 with varied pH conditions.

electron-withdrawing or electron-donating group. c1 and c2 show different spectra phenomena under different pH conditions because of the protonation and deprotonation of the nitrogen atoms. The resultant BODIPYs can be appropriately applied as cell imaging materials due to their good physical chemical characteristics and excellent biocompatibility. For c2 in mixed solvents of ethanol and glycerol with different proportions, the logarithm of the fluorescence intensity increased linearly with the logarithm of the solvent viscosity, highlighting the potential of c2 to be applied to test the intracellular viscosity changes of the living cell for disease diagnosis.

4. Conclusion Two new BODIPY derivatives containing both BODIPY fluorophores and the indole moieties with NO2 and NH2 groups have been synthesized and characterized by NMR, FTIR and HRMS. The UV–vis and PL spectra results of the prepared BODIPY compounds verify that the electron-withdrawing or electron-donating effect and negative solvatochromism affect their molecular excited states in various solvents and led to the difference in absorption and emission behaviors. Theoretical calculations and results of CV and photostability approve that the oxidation potential and stability of c1 and c2 could be tuned by the

Fig. 10. (a)Effect of different viscosity of mixed solvents on fluorescence intensity of c2. (b) A linear relationship between logarithm of emission intensity and the solvent viscosity. 163

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Acknowledgements

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