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Study on the pH-Dependence of the Photophysical Properties of a Functionalized Perylene Bisimide and Its Potential Applications as a Fluorescence Lifetime-Based pH Probe Pedro J. Pacheco-Liñán, Mónica Moral, María L. Nueda, Rubén Cruz-Sánchez, Jesús FernándezSainz, Andrés Garzón-Ruiz, Ivan Bravo, Manuel Melguizo, Jorge Laborda, and Jose Albaladejo J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.7b07839 • Publication Date (Web): 10 Oct 2017 Downloaded from http://pubs.acs.org on October 15, 2017

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The Journal of Physical Chemistry

Study on the pH Dependence of the Photophysical Properties of a Functionalized Perylene Bisimide and Its Potential Applications as a Fluorescence Lifetime-Based pH Probe

Pedro J. Pacheco-Liñán†; Mónica Moral‡; María L. Nueda§; Rubén Cruz-Sánchez∥; Jesús Fernández-Sainz†; Andrés Garzón-Ruiz*,†; Iván Bravo†; Manuel Melguizo∥; Jorge Laborda§ and José Albaladejo⊥

†

Departamento de Química Física, Facultad de Farmacia, Universidad de Castilla-La Mancha,

Paseo de los Estudiantes, s/n, 02071 Albacete, Spain. E-mail: [email protected] ‡

Instituto de Investigación de Energía Renovables, Universidad de Castilla-La Mancha, Paseo de la

Investigación 1, 02071 Albacete, Spain §

Departamento de Química Inorgánica, Orgánica y Bioquímica, Facultad de Farmacia, Universidad

de Castilla-La Mancha, Paseo de los Estudiantes, s/n, 02071 Albacete, Spain. ∥

Departamento de Química Inorgánica y Orgánica, Facultad de Ciencias Experimentales,

Universidad de Jaén, Campus las Lagunillas, 23071 Jaén, Spain ⊥

Departamento de Química Física, Facultad de Ciencias Químicas, Universidad de Castilla-La

Mancha, Avenida Camilo José Cela, 10, 13071 Ciudad Real, Spain.

ABSTRACT In this work, we have investigated the dependence on the pH of the photophysical properties of a functionalized perylene bisimide (PBI) and its potential pH sensing applications. It was observed the presence of aggregates which diminishes at acid pH values and low concentrations, without totally disappearing until a temperature of 80ºC was reached. At basic pH, significant changes in the absorption spectrum were observed, which were associated to more strongly coupled aggregates. The 1H-NMR spectra of the PBI dye in D2O/TFA also showed the dependence of aggregation on 1 ACS Paragon Plus Environment


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concentration and temperature. PBI fluorescence intensity and lifetime were also sensitive to pH values. The maximum fluorescence intensity and lifetime were observed in acid medium, in which protonation of the secondary amines on the PBI side chains likely hinders the formation of strongly coupled aggregates. On the contrary, the fluorescence intensity significantly decreased in basic medium, due to deprotonation of the amine groups and the formation of stronger aggregates. Density Functional Theory calculations corroborated that π-stacked aggregates of PBI derivatives are stable in the protonated state, but their supramolecular structure changes. In the aggregate, monomeric units slide over their adjacent ones and increase the intermolecular distance upon the protonation. Intermolecular hydrogen bonds can help maintaining the stability of the protonated aggregate. Fluorescence lifetime showed a sigmoidal dependence on pH, with a linear response range between pH 6 and 8, both in Tris-HCl buffered solutions and in a synthetic buffer mimicking the intracellular environment. The biocompatibility of the PBI dye was tested in C3H10T1/2 mesenchymal cells. The cellular uptake was confirmed by confocal fluorescence microscopy. No significant effects on cellular viability and morphology were observed at the conditions in which compound 1 can be used as a fluorescent probe. This work supports the idea that PBI derivatives can be suitable dyes for fluorescence lifetime sensing applications.

INTRODUCTION Perylene bisimides (PBIs) are prototypical molecules frequently used in fundamental spectroscopic studies, such as molecular aggregation1,2. A wide range of applications in chemistry, biology and materials science has been reported for these compounds. For example, they are commonly used as fluorescence-based sensors and imaging3–8, in the fabrication of organic electronic devices9–11, and as scaffolds in supramolecular2,12 and

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in metallosupramolecular9 architectures. The suitability of PBIs for such diverse applications is due to their molecular structure: a rigid polycyclic aromatic scaffold perylene substituted with electron-withdrawing imide groups1 . In this work, we aimed to study the use of PBI dyes as pH sensors, specifically as pH-dependent fluorescence lifetime-based sensors. Different fluorescent conjugated molecules are being investigated as indicators of abnormal intracellular pH values associated with different disorders, such as cancer and Alzheimer’s disease13–15. In particular, functionalized molecules and polymers based on tristyrylbenzene16 , diketopyrrolopyrrole17–19, BODIPY20–22, naphthalene bisimide23,24 and PBI4,5,25 have been studied as pH-sensitive fluorescent sensors. Nevertheless, only a small number of works have focused on the pH dependence of the fluorescence lifetime of conjugated organic dyes. Fluorescence lifetime sensors are particularly promising for biological imaging techniques, since lifetime does not depend on fluorophore concentration, fluorescence intensity, excitation wavelength and duration of light exposure26–29. To the best of our knowledge, only a single work has explored the behavior of a pH-sensitive probe based on PBI in live cells by means of fluorescence lifetime imaging microscopy (FLIM)25. In the present work, we have investigated the fluorescence intensity and lifetime as a function of pH for a molecule with a PBI core functionalized with N-hydroxyethylaminoethyl chains at the imide positions (see Chart 1). The fluorescence response to pH variation was related to the molecular aggregation of the PBI cores and the photoinduced electron transfer (PET) effect due, in turn, to the protonation-deprotonation processes of the amine groups in the side chains. The influence of amine group protonation on the molecular aggregation of 1 has also been investigated from a theoretical standpoint using Density Functional Theory (DFT) calculations. Finally, we



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have studied the toxicity and uptake of 1 in C3H10T1/2 cells to get new insights on the feasible use of PBI dyes as fluorescence lifetime-based pH probes in living cells. HO O

O

N

N

O

O

NH NH OH

Chart 1. Chemical structure of the compound 1

MATERIALS AND METHODS Materials. The reagents, perylene-3,4,9,10-tetracarboxylic acid dianhydride (97 %) and 2[(2-aminoethyl)amino]ethanol (99 %), as well as toluene (for analysis) and hydrochloric acid (reagent grade, 37 %) consumed in the synthesis of 1 were purchased from Sigma-Aldrich and used without further purification. Ficoll®400, bovine serum albumin (BSA), Tris-HCl buffer, different inorganic salts (NaCl, KCl, MgSO4 and CaCl2) and chloroform employed in spectroscopic studies were purchased from Sigma-Aldrich. Thiazolyl blue tetrazolium bromide (MTT) was acquired from Acros. Dimethyl sulfoxide (DMSO) and trifluoroacetic acid (TFA) was provided by VWR. Dulbecco’s modified Eagle’s medium (DMEM), DMEM without phenol red, fetal bovine serum (FBS), penicillin-streptomycin and L-Glutamine were purchased from Lonza. All aqueous solutions were prepared in Milli-Q water and filtered with 0.22 µm filters prior to use. The pH of the aqueous solutions and buffers was adjusted using NaOH or HCl. All reagents used were of molecular biology grade. Stock solutions were kept at 4ºC in the dark. Synthesis

of

N,N’-bis{2-[(2-hydroxyethyl)amino]ethyl}perylene-3,4,9,10-

tetracarboxybisimide, 1. To a suspension of perylene-3,4,9,10-tetracarboxylic acid 4 ACS Paragon Plus Environment

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dianhydride (0.25 g, 6.38 mmol) in toluene (10.0 mL), 2 mL (1.67 g, 15.95 mmol) of 2[(2-aminoethyl)amino]ethanol were added and the resulting mixture was refluxed for 24 h with continuous stirring under Ar atm. The dark solid in suspension was collected by filtration, washed with toluene, and dried in the pump. Then, it was transferred to a beaker and stirred with 1N aqueous HCl (100 mL) at room temperature for 15 min. The resulting solid was collected by filtration, washed with 1N aqueous HCl and dried in the pump to afford a dark red crystalline solid identified as the dihydrochloride of the title compound, 1·2HCl (0.39 g, 0.61 mmol, 95 %). 1H NMR (400MHz, D2O/TFA, 5:1, v/v, 353 K) δ = 7.90 (d, J = 8.0 Hz, 4 H), 7.59 (d, J = 8.0 Hz, 4 H), 4.46 (t, J = 6.5 Hz, 4 H), 3.98 (t, J = 5.3 Hz, 4 H), 3.57 (t, J = 6.6 Hz, 4 H), 3.42 (t, J = 5.3 Hz, 4 H) ppm30. 1H RMN (D2O) δ = 7.63 (br s, 4H), 7.26 (br s, 4H), 4.31 (br s, 4H), 3.87 (br s, 4H), 3.42 (br s, 4H), 3.30 (br s, 4H) ppm. 13C-RMN (126 MHz, SS-MAS-CP/TOSS) δ = 165.9, 164.7, 134.4, 125.1, 59.2, 52.6, 47.6, 39.5, 36.6 ppm. FTIR (ATR) ν: 742.57, 811.88, 866.86, 1019.80, 1071.80, 1170, 1250.80, 134.3020, 1401, 1447.20, 1597.40, 1649.30, 1695.50, 2781.40, 2966.20, 3500 cm-1. HRMS (Q-TOF, ESI) m/z: (M+H)+ Cald. for C32H29N4O6: 565.2082, found: 565.2080; (M+2H)+2 Cald. for C32H30N4O6: 283.1077, found 283.1078. Anal. Calcd. for C32H30N4O6·2HCl: C, 60.29; H, 4.74; N, 8.79; Found: C, 60.07; H, 4.71; N, 8.64. Structural characterization. Structural characterization of compound 1 was performed in CICT-Universidad de Jaén.

1

H-NMR spectra of compound 1 in solution were

acquired in a Bruker Avance 400 instrument, operating at 400 MHz and equipped with a BBO probehead. NMR solvents were commercial deuterium oxide (99.9 %D) and trifluoracetic acid (TFA, 99 %) from Sigma-Aldrich. Solid-state 13C-MNR spectra were acquired in a Bruker Avance 500 equipped with a standard bore 11.7 T superconducting



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magnet, operating at 500 MHz (1H), by means of a Bruker dual channel magic angle spinning (MAS) broadband probehead for solid samples. The FTIR spectrum of pure solid 1·2 HCl was acquired in a BRUKER TENSOR 27 TGA-IR by means of a BRUKER Platinum ATR system incorporating a diamond crystal. High-resolution mass spectra (HRMS) of 1 were recorded in a time-of-flight instrument Micromass LCT Premier from Waters, connected to a high resolution liquid chromatograph (HPLC), Alliance 2795 from Waters, and were obtained by electrospray ionization (ESI). Combustion elemental microanalyses were performed in a Thermo Finnigan Flash EA 1112 series equipment. Absorption and Fluorescence Measurements. UV-Vis absorption spectra were acquired on a Cary 100 (Varian) spectrophotometer at room temperature, using a slit width of 0.4 nm and scan rate of 600 nm min-1. Steady state fluorescence (SSF) and time

resolved

fluorescence

(TRF)

spectra

were

recorded

on

an

FLS920

spectrofluorometer (Edinburgh Instruments) equipped with a time correlated single photon counting (TCSPC) detector. A TLC 50 temperature-controlled cuvette holder (Quantum Northwest) was used for the measurements (temperature was kept constant at 298 K excepting for the temperature-dependence experiments). Ten mm quartz cuvettes (Hellma Analytics) were employed for all spectroscopic measurements. For SSF spectra, a 450 W Xe lamp was used, and the light source and the excitation and emission slits were both fixed at 1 nm, except where otherwise expressly indicated. The step and dwell time were 1 nm and 0.1 s, respectively. For TRF experiments, an EPLED 560 sub-nanosecond pulsed light emitting diode (Edinburgh Photonics) was employed as light source at 565 nm, and the fluorescence decay profiles were collected at 590 nm. The fluorescence intensity decays, I(t), were fitted by using an iterative least squares fit method to the following multiexponential function:


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= ∑ exp−/

(1)

where αi and τi are the amplitude and lifetime for each ith term. The average decay lifetime was then calculated as: =

∑

(2)

∑

Two polarizers placed in the excitation and emission light pathways were employed for steady-state fluorescence anisotropy measurements. Polarized emission spectra were recorded at four polarizer directions, i.e. both horizontal (HH), both vertical (VV), horizontal on excitation and vertical on emission (HV), and vice versa (VH). The anisotropy spectra were calculated as: !! " !# !! $ % !#

=

(3)

where Ii represents the fluorescence intensity at the corresponding configuration of the polarizers and G is a correction factor given by: &=

#!

(4)

##

A sample concentration of 10 µM was generally employed for UV-Vis absorption spectroscopy experiments and a concentration of 1 µM for fluorescence assays, except where otherwise expressly indicated. For pH dependence experiments, sample aqueous solutions buffered with Tris-HCl (10 mM) were titrated by successive additions of small volumes (in the order of microliters) of HCl or NaOH solutions at different concentrations (0.01 – 10 M). In addition, a synthetic buffer (SIB) mimicking the intracellular environment [10 mM Tris-HCl, 150 mM K+, 50 mM Na+, 1 mM Ca2+, 1 mM Mg2+, 1% Ficoll®400, and 0.2 mg mL-1 bovine serum albumin (BSA)] was used to test the behavior of 1 as a pH-sensitive fluorescence lifetime molecular probe in a biological medium31. To examine the effect of concentration on the aggregation, a titration was performed with a concentrated solution of 1 within the range 0.2 – 50 µM.



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Chloroform solutions of 1 were also titrated with trifluoroacetic acid to assess whether the fluorescence signal was also quenched by the PET effect. Quantum yields were measured in a FS5 spectrofluorometer (Edinburgh Instruments) equipped with an integrating sphere, a 150 W Xe lamp as the light source and a TCSPC detector. For these measures, the excitation wavelength, excitation and emission slits, step and dwell time were fixed at 480 nm, 10 nm and 0.14 nm, 1 nm and 0.5 s, respectively. Fluorescence reabsorption was reduced by dilution of the sample (1 µM) and corrected by using the equation: Φ()*+ =

,-./ " $

0 1-./ 22

(5)

where Φtrue and Φobs correspond to the true (corrected) and observed (directly measured) quantum yield, respectively. The reabsorbed area, a, is obtained as the quotient of the emission spectra areas, as measured in the integrating sphere, and the standard sample cuvette holder.

Cell culture. C3H10T1/2 cells (clone 8, ATCC® CCL-226™) were grown by incubation in DMEM containing 10% FBS, 5% glutamine and 5% penicillin/streptomycin, at 37°C in a 5% CO2 humidified atmosphere, as described previously32. To evaluate the uptake and intracellular localization of the compound 1, cells were cultured on six-well plates. When cells reached 50% of confluence, they were treated with a 0.5 µM aqueous solution of 1 for 1h. Then, the cells were washed three times with PBS and laserscanning confocal images were taken with a Zeiss LSM710 confocal microscope. Compound 1 was excited with the 488 nm line of Ar ion laser and the fluorescent emission was recorded from 502 to 552 nm (green channel). EC PlanNeofluar 40x/0.75 M27 objective was used. Transmitted light detector (T-PMT) was used by bright field images. 8 ACS Paragon Plus Environment

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Viability assay. Cell viability was measured by MTT assays according to the manufacturer´s protocol (Sigma). Ten thousand cells were seeded per well in a 96-well plate, and incubated for 24h. Then, cells were incubated for 24h with 0.25 to 5.0 µM concentrations of compound 1 in DMEM without phenol red. Afterwards, cells were incubated for another 45 min. with 100 µL of a MTT solution (5 mg mL-1) in DMEM without phenol red. At the end of the incubation period, the medium was removed and the reduced dye was solubilized with DMSO. Finally, the absorbance at 570 nm was measured in an ELISA 96-well plate using a plate reader spectrophotometer (Ez Read 400, Biochrom). The viability of the treated cells was determined for each concentration by comparing their absorbance values with those of the untreated control cells. Three independent experiments were performed for each concentration of compound 1 examined. Results were represented as the means ±SD of different experiments. Computational details. The Gussian09 package (revision D.01)33 was employed for all calculations. The DFT hybrid functional B3LYP34,35, together with the Pople’s basis set 6-31G*, were used for the computation of the molecular structure and electronic properties of compound 1, both as a free molecule and in aggregate state. Geometry optimizations were performed both in the gas phase and in aqueous solution, where the solvent was described by the Polarizable Continuum Model (PCM)36. Moreover, we have introduced the non-covalent intermolecular interactions to the hybrid functional in the calculations of the molecular aggregate by using the empirical D3(BJ) dispersion correction for DFT, as proposed by Grimme et al.37,38. This way of considering the noncovalent energy has been revealed as an efficient and accurate method39. Finding the most energetically favorable molecular arrangement in the aggregated state is generally a complex task due to the large number of local minima existing on the potential energy hypersurface. For that reason, we performed a careful analysis on



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the dependence of binding energy with the azimuthal angle (α) and (x,y)-displacement of one molecule with respect to another in molecular dimers, as shown in Figure 1. Initially, four dimers were built with the previously optimized monomer structure at α = 0°, 30°, 60° and 90°. Then, the binding energy was calculated for each dimer as a function of the relative (x,y)-displacement between both molecules. For each starting structure at α = 0°, 30°, 60° and 90°, a large number of dimers were then generated through (x,y)-displacements between 5.0 and 14.0 Å, with a grid size of 0.5 Å, and a zdisplacement kept fixed at 3.5 Å, the typical π-stacking distance40. Binding energy was calculated for each dimer —including D3(BJ) correction without further optimization— as the energy difference between the dimer and the isolated monomers, and plotted as a function of the relative (x,y)-displacement. Once a global minimum was localized on each energy landscape, these molecular arrangements were used for the optimization of the corresponding dimers and tetramers. d1, d2, d3 and d4 correspond to the optimized dimers extracted from the energy landscape with α = 0°, 30°, 60° and 90°, respectively. Here, it must be noted that after geometry optimization, the initial α values of the molecular aggregates can be modified. The geometry optimization of the molecular tetramers was accomplished using the ONION methodology41. Thus, two layers were defined: the high accuracy layer, involving the two central molecules (computed at the B3LYP-D3(BJ)/6-31G* level), and the low accuracy layer, corresponding to the terminal molecules, (computed at the B3LYP/6-21G* level). A similar notation was employed for the molecular tetramers, t1, t2, t3 and t4. A structural optimization of the molecular dimers was accomplished, both in a gas phase and in aqueous solution, to assess the effect of the solvent on the geometry of the molecular aggregates (the tetramers were only optimized in aqueous solution).


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z-axis

y-axis

HO NH

O N O

O N O

x-axis

NH OH

O

HO

N NH

O

O

α

NH OH

N O

Figure 1. Two stacked molecules showing the azimuthal angle (α) and (x,y)displacement directions. (z)-distance was kept fixed at 3.5 Å

In a second step, we aimed to determine the effect of protonation on the molecular structure of the aggregates. For that, molecular dimers, d1, d2, d3 and d4, were optimized again in diverse protonation states. Figure 2 shows the four different protonation states considered for the dimers, i.e. state 0 (neutral molecule), +2 (diprotonated molecule in two different conformations, i.e. +2a and +2b), and +4 (fully protonated molecule). Optimization of molecular tetramers requires large consumption of computational resources and hence only the neutral and fully protonated states were calculated.



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HO

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HO

NH

HO NH

O

O

N

N

O

O

O

O

N

N

O

O

+

NH2

NH HO

OH

+

NH2

NH

O

O

N

N

O

O

O

O

N

N

O

O

NH OH

NH OH

OH

State 0

State +2a

HO

HO +

NH2

HO NH

O

O

N

N

O

O

O

O

N

N

O

O

+

NH2 NH HO OH

+

NH2 +

NH2

O

O N

N

O

O

O

O

N

N

O

O

+

NH2

OH +

NH2

OH

OH

State +2b

State +4

Figure 2. Scheme of the different protonation states considered for the molecular dimers.

RESULTS AND DISCUSSION Photophysical studies. Compound 1 is soluble in water and its hydrophilic character is caused, in part, by protonation of the amine groups in the N-hydroxyethyl-aminoethyl side chains. Figure 3 shows the absorbance spectrum of compound 1 in a 10 µM aqueous solution. The maximum absorption wavelength (λabmax) associated to the S0→S1 electronic transition appeared at 500 nm, along with a peak centered at 535 nm and a blue-shifted shoulder. The characteristic vibronic structure of that band is not clearly defined and this behavior is commonly attributed to π-π aggregation of PBI cores1,2,12,42. The blue-shift observed in the spectrum with respect to that expected for the monomer suggests the formation of cofacial aggregates1,2. The effect of the concentration on the molecular aggregation of 1 was investigated through the ratio of absorptions between S0(v=0) → S1(v´=0) and S0(v=0) → S1(v´=1) vibronic transitions, noted as A0-0 and A0-1, respectively. In PBI dyes, A0-0/A0-1 ratios near 1.6 indicate 12 ACS Paragon Plus Environment

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absence of aggregation, whereas lower values are related to the formation of aggregates. As expected, the A0-0/A0-1 ratio —recorded for 1 in buffered aqueous solutions (pH = 7.4)— increases with dilution (see Figure 4). However, values of A0-0/A0-1 < 1.0 were observed for concentrations as low as 0.25 µM, indicating the presence of aggregates even at these low concentrations10,12,43. The aggregation state of 1 also depends on the pH of the medium due to the presence of protonable amine groups at the side chains. In acid medium, compound 1 at a concentration of 10 µM is not fully disaggregated, since A0-0/A0-1 amounts to 0.50 at pH 2.0. At basic pH values, the absorption spectrum undergoes dramatic changes, indicating strong π-π interactions. The significant loss of intensity and blue shift observed in the absorption band can be related to the formation of cofacial (or near-cofacial) aggregates (see Figure 5a). For 1 µM solutions in acid medium (pH = 1.3), nearly total disaggregation of 1 was achieved at a temperature of 80ºC (A0-0/A0-1 = 1.55; see Figure 5b), whilst aggregates at basic pH showed significantly higher stability (see Figure S7).

1.00 Excitation Emision Absortion

0.75

0.75

0.50

0.50

0.25

0.25

0.00 400

500

600

Normalized fluorescence

1.00

Normalized absorbance

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0.00 700

Wavelength (nm) Figure 3. Normalized absorption, excitation and fluorescence spectra of a 10 µM compound 1 solution in water (pH = 6.0 and T = 298 K). λex = 495 nm and λem = 547 nm were used to record the emission and excitation spectra (the slit widths were ∆λex = 1 nm; ∆λem = 4 nm), respectively. 13 ACS Paragon Plus Environment


(a)

1.0 0.9

A0-0/A0-1

0.8 0.7 0.6 0.5 0.4 0.1

1

10

100

[1] (µM) (b)

0.2 µM 0.5 µM 1.0 µM 2.5 µM 5.0 µM 10.0 µM 25.0 µM 50.0 µM 75.0 µM

5

4

Fluorescence intensity (× 10 a.u.)

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

(1) (2)

4 3 2 1 0 500

550

600

650

700

750

Wavelength (nm) Figure 4. (a) A0-0/A0-1 ratios measured in the absorption spectra at concentrations ranging from 0.2 µM to 75.0 µM; (b) Emission spectra recorded at the different concentrations indicated. Both experiments were carried out in aqueous solution buffered with Tris/HCl (pH = 7.4, λex = 495 nm and T = 293 K).


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(a)

0.25

pH 2.1 pH 5.1 pH 6.1 pH 7.0 pH 8.0 pH 9.0 pH 11.0

Absorbance

0.20 0.15 0.10 0.05 0.00 400

450

500

550

600

650

Wavelength (nm) (b)

0.06

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


10ºC 30ºC 50ºC 80ºC

0.04

0.02

0.00 400

500

600

700

Wavelength (nm) Figure 5. (a) Absorption spectra of a 10 µM solution of compound 1 at the different pH values indicated; (b) Absorption spectra of a 1 µM solution of compound 1 in a HCl aqueous solution (pH = 1.3) at the different temperatures indicated. The 1H-NMR spectra of 1 in D2O/TFA (5:1, v/v) also reflects an aggregation process dependent on concentration and temperature. Thus, the NMR signals of the aromatic



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protons of the perylene units of 1 registered at 300 K suffer a progressive deshielding (of 0.30 - 0.40 ppm) after dilution from 7.8 mM to 5.0 mM (see Figure S2, in Supporting Information). In aggregations produced by π-π cofacial interaction between aromatic rings, the NMR chemical shift of the hydrogen atoms directly bonded to the aromatic rings is sensitive to the extent of the aggregation process44,45. This is due to each aromatic unit that take part in an aggregate lies in the NMR magnetic shielding cones of the aggregate’s adjacent aromatic molecules. Consequently, the mean NMR shielding suffered by the hydrogen atoms bonded to the aromatic rings would be more pronounced as the proportion of molecules involved in the aggregates become larger. Thence, the 1H-NMR deshielding observed upon dilution in our molecules is coherent with the lessening of a π-stacking aggregation process between the perylenebisimide molecules. Similarly, an increase in temperature would produce a shifting of the aggregation/solution balance towards a higher proportion of molecules in solution that will also manifest in the 1H-NMR signals of the perylene hydrogen atoms. In our experiment on NMR spectra taken from a 5.0 mM solution of 1 in D2O/TFA (5:1, v/v) at different temperatures from 300 to 353 K (see Figure S3), a further deshielding (of 0.45-0.60 ppm), accompanied by a definition of the homonuclear coupling features of that signals (becoming well resolved doublets with H-H coupling constants J = 8.0 Hz), was clearly observed, so confirming the predominance of individual molecules of 1 at 353 K in the 5.0 mM acidic solution. Figure 3 shows the fluorescence excitation and emission spectra of 1 in aqueous solution. In contrast with the absorption spectra recorded at room temperature, the excitation and emission spectra of 1 do exhibit the characteristic vibronic structure of non-aggregated PBI dyes. That fact is consistent with the pronounced emission


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quenching that is observed in cofacial aggregates, i.e. strongly aggregated species must have a low contribution to the fluorescence emission1,2,46. In acidic solutions, the protonation of the amine groups hinders the formation of strongly coupled aggregates in some way, and reduces the fluorescence quenching effect30,42. As shown in Figure 6a, the fluorescence emission intensity decreases with increasing pH values, but no significant changes in the spectrum shape occur. Quantum yields in parallel decrease from Φ = 16.7% to Φ = 1.8% and Φ = 0.5% for pH values of 5.2, 7.4 and 10.2, respectively. For its part, steady-state fluorescence anisotropy increases with the aggregation, from r = 0.003 for pH 3.1 to r = 0.013 for pH 10.0 (see Figure S8). Hence, the change in fluorescence emission intensity as a function of the pH seems to be related to the evolution of the aggregation state as a consequence of the protonation state of the amine groups. However, we cannot discard that the intense quenching observed at basic pH values is also caused by photo-induced electron transfer (PET) from the deprotonated side-chain amines. This phenomenon has already been observed in PBI dyes with secondary and tertiary amines linked to the imide groups through two carbon chains25,42. Figure 6b shows the increase in fluorescence intensity of a 1 µM solution of 1 in chloroform upon titration with trifluoroacetic acid (TFA). Chloroform is a good solvent typically used to disaggregate PBI dyes in solution.12,42 The increase in the fluorescence intensity with the addition of TFA can be attributed to the PET phenomenon, since the solvent hinders the aggregation process and fluorescence quenching becomes less efficient12,42.



4

pH 2.1 3.3 4.1 6.0 7.1 8.3

5

Fluorescence intensity (×10 a.u.)

(a)

3

2

1

0

550

600

650

700

750

Wavelength (nm)

(b)

[TFA]:[1]

1.2

0.00 0.01 0.10 1.00 2 4 8 10 20 50 100 500 1000 10000

5

Fluorescence intensity (×10 a.u.)

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

1.0 0.8 0.6 0.4 0.2 0.0

550

600

650

700

Wavelength (nm)

Figure 6. (a) Steady-state fluorescence emission spectra of a 1 µM aqueous solution of compound 1 at different pH values (λex = 495 nm); (b) Steady-state fluorescence emission spectra of a 1 µM solution of compound 1 in chloroform titrated with trifluoroacetic acid (TFA) (λex = 495 nm).


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Theoretical Study of the Molecular Aggregation. Figure 7 shows the binding energy landscapes computed for stacked dimers, with different azimuthal angles (α = 0º, 30º, 60º and 90º), as a function of the relative (x,y)-displacement. The binding energy minima (in black color on Figure 7) represents the dimers most strongly coupled. The global minimum appears in the energy landscape of α = 30º (EB < -30.0 kcal mol-1) for x- and y-displacements lower than 2.0 and 0.5 Å, respectively. On the contrary, the highest energy was computed for the maximum overlap configuration, concluding that nuclear repulsion dominates the binding energy landscape in this region (see the plot corresponding to α = 0º in Figure 7). For α = 60º and 90º, broad minima were found for x- and y-displacements lower than 4.0 and 1.0 Å, respectively. Then, four molecular dimers, d1 – d4, were optimized using as a starting point the global minima obtained from the four binding energy landscapes shown in Figure 7. Table 1 collects the relative energies calculated in aqueous solution for dimers d1 – d4 in different protonation states (see Figure 2), along with some supramolecular structural parameters which will help to analyze the evolution of the molecular aggregate with the pH changes of the medium (additional information on the calculations in the gas phase can be found in Table S1, in Supporting Information). According to calculations in aqueous solution, d2 is the most energetically favorable molecular dimer in deprotonated (0) and diprotonated states (+2a, +2b), whereas d1 corresponds to the most favorable molecular arrangement when the system is fully protonated (+4) (the optimized molecular arrangements of d2 (state 0) and d1 (state +4) states are shown in Figure 8). All these four dimers show dihedral angles |φ| ranging from 33.6º to 36.8º (the notation used for these supramolecular parameters can be consulted in Figure 9). It is worth mentioning that d1 in the deprotonated state shows a near cofacial arrangement (θ = 72.9º and |φ| = 3.0º), but the system escapes from the local minimum when is re-optimized in state +4 and adopts a



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substantially different arrangement (θ = 124.1º and |φ| = 33.6º). Angle θ allows us to estimate the sliding between the dimer molecules when the protonation state changes. Thus, considering the two global minima in the deprotonated state, d2 (0), and in the fully protonated state, d1 (+4), it can be seen that the angle θ increases from 109.3º to 124.1º, which indicates sliding of one molecule over the other, at the same time that the dihedral angle |φ| is maintained near the value of 35º. This molecular sliding is also reflected by the change in the intermolecular distance, dct, with protonation (dct varies from 3.64 Å for d2 (0) to 4.32 Å for d1 (+4)). In addition to the sliding, the separation between the planes involving both molecules, i.e. dπ, which was defined as the minimum distance between the cores of the two stacked molecules, increases from 3.20 Å for d2 (0) to 3.48 Å for d1 (+4). Intermolecular hydrogen bonds involving the sidechain amine groups and core carbonyl groups were calculated in some aggregates (see Figure 8). These potential interactions would help to maintain the stability of these aggregates in the protonated states. These results are consistent with the experimental observations, i.e. at acidic pH, PBI molecules remain aggregated, but the molecular coupling is weaker than in an alkaline medium, inducing a higher fluorescence emission intensity. Calculations in the gas phase indicated that d2 is the most energetically favorable dimer in the 0 and +4 states and it was also observed a separation of the molecules when the dimer is fully protonated. The dihedral angle |φ| of d2 (0) in the gas phase is similar to that computed in aqueous solution, but |φ| increases up to 51.3º in the fully protonated state, reflecting the strong effect of the solvent on the geometry of the molecular aggregate. To minimize edge effects, molecular tetramers were also modeled in aqueous solution. In these conditions, t3 and t4 are the most energetically favorable molecular aggregates in deprotonated (0) and fully protonated (+8) states, respectively. Considering only the central molecules of those tetramers, |φ2| values (falling within 34


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– 45º) are comparable to those observed for the previously modeled molecular dimers. Again, a clear increase in molecular separation was observed upon protonation (3.44 Å to 4.01 Å for dct2, and 3.26 Å to 4.19 Å for dπ2).

5

5 -30.0 kcal/mol

-5.000 kcal/mol

y-displacement / Å

y-displacement / Å

-30.00 kcal/mol

20.00 kcal/mol

4

75.00 kcal/mol 122.5 kcal/mol 155.0 kcal/mol

3

187.5 kcal/mol 220.0 kcal/mol

2

1

0 0

2

4

6

8

10

12

0 kcal/mol 20.0 kcal/mol

4

40.0 kcal/mol 60.0 kcal/mol 80.0 kcal/mol

3

100 kcal/mol 110 kcal/mol

2

1

0

14

0

2

x-displacement / Å

4

6

8

10

x-displacement / Å

α = 0º

α = 30º -29 kcal/mol

5

-10 kcal/mol

4

0 kcal/mol 5.0 kcal/mol 9.0 kcal/mol

3

15 kcal/mol

2

1

0 1

2

3

4

5

6

7

-26 kcal/mol

5

-20 kcal/mol

y-displacement / Å

y-displacement / Å

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


-22 kcal/mol -18 kcal/mol -14 kcal/mol

4

-10 kcal/mol -5.0 kcal/mol 0 kcal/mol

3

5.0 kcal/mol

2

1

0

8

0

x-displacement / Å

1

2

3

4

5

6

7

x-displacement / Å

α = 60º

α = 90º

Figure 7. Binding energy (in kcal mol-1) landscapes calculated at the B3LYP/6-31G* level of theory for dimers with different azimuthal angles (α) and a stacking distance of z = 3.5 Å.



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

d2 (state 0)

d1 (state +4)

t3 (state 0)

t4 (state +8)

Figure 8. Optimized molecular arrangements of the most energetically favorable molecular dimers and tetramers in deprotonated and fully protonated states in aqueous solution.


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(a) HO O

NH

O ct1

N O

HO

N θ

NH

O

dct

OH

O

NH

O

N

N

ct2

O

NH

O

dct = d(ct1-ct2)

OH

θ = τ(N1-ct1-ct2) φ = τ (N1-ct1-ct2-N2)

(b) HO NH

O

O

ct1

N

N

O

HO

NH

O

OH O

NH

O

ct2

N

N

O

HO

NH

O

OH O

NH

O

ct3

N

N

O

HO

NH

NH

O

O

O

N

OH

N

ct4

O

O

NH OH

dct1 = d(ct1-ct2)

θ1 = τ(N1-ct1-ct2)

φ1 = τ(N1-ct1-ct2-N2)

dct2 = d(ct2-ct3)

θ2 = τ(N2-ct2-ct3)

φ2 = τ(N2-ct2-ct3-N3)

dct3 = d(ct3-ct4)

θ3 = τ(N3-ct3-ct4)

φ3 = τ(N3-ct3-ct4-N4)

Figure 9. Notation of the supramolecular structural parameters used in our discussion (ct is the abbreviation for centroid). 23 ACS Paragon Plus Environment


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Table 1. Relative energy (ER) calculated in aqueous solution for the molecular dimers d1 – d4 in different protonation states. In each protonation state, the lowestenergy dimer was considered as the reference structure for ER calculation. Supramolecular structural parameters of the optimized molecular dimers are indicated. dπ corresponds to the minimum distance between the cores of the two stacked molecules, dct is the distance between two centroids, θ corresponds to the angle between N1-ct1-ct2, and φ is the dihedral angle between N1-ct1-ct2-N2 (see Figure 9). Di mer d1

Sta te 0 +2

ER / kcal mol-1 4.44

0 +2

0.00

b

22 +4 0 +2

78

+2

3.

11.33

3.

0 +2 a/b a +4

17.83

3. 3. 3.

a

93.23

36.81

4.

100.09

40.75

3.

66.03

34.78

3.

112.57

40.78

3.

112.57

39.71

4.

100.60

40.58

3.

81.03

54.84

3.

111.65

41.32

3.

95.29

41.92

55 3.

44

3.

56

34 11.72

34.36

05

25 19.53

98.84

84

23 21.90

3.

78

24 +4

36.61

71

30

b

d4

3.

6.35

109.36

15

22

a

3.

42 3.

5.96

33.62

43 3.

19.82

124.11

4.

3.

0.00

2.35

/

64

21 +2

71.73

32 3.

0.00

1.60

3.

3.

20

a

72.12

94

48 0.00

3.03

3.

3.

|φ| degrees

72.92

93

22 +4

3.

3.

8.10

θ / degrees

75

29 +2

d3

3.

2.51

b

dct /Å

32

a

d2

dπ /Å

53

The starting point for the optimization of d4 in +2a and +2b states are equivalents.

pH-Sensitive Fluorescence Lifetime Probe. In addition to fluorescence emission intensity, the fluorescence lifetime of compound 1 is also sensitive to the pH of the solution (see Figure 10). In our experiments, we observed a ten-fold decrease of the 24 ACS Paragon Plus Environment

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fluorescence average lifetime, τav, from pH 5.2 to 10.2. Nevertheless, no significant influence of the concentration on the dye fluorescence lifetime was observed within the range 0.5 – 5.0 µM (see Table 2). Figure 11 shows the fluorescence lifetime dependence of 1 (1 µM) with the pH, which evolves as a classical S-shape titration curve with a linear response range between pH 6 and 8 (pKa = 7.0). This behavior could be interesting for biosensing applications, considering the variation range of the fluorescence lifetime includes the physiological pH range, and it is independent of the concentration. For that reason, a synthetic intracellular buffer (SIB), including proteins, polysaccharides, and inorganic salts, was employed to test the fluorescence response of 1 to pH variations in a medium mimicking the intracellular environment. The solution of 1 in SIB brought out a significant decrease of the maximum τav (for pH < 6.0) from ~ 4.2 ns (in Tris-HCl buffer) to ~ 2.5 ns (in SIB buffer). This variation of the fluorescence lifetime could be attributed to two factors: (i) a modification of the aggregation state of 1 due to the increase of the solvent polarity by the presence of salts47 (ii) an enhancement of the fluorescence quenching by macromolecules such as BSA and Ficoll. Despite this decrease in the fluorescence lifetime at acid pH values, τav keeps a linear relationship with pH within the range of 6.0 to 8.0. The τav change (~2 ns) and pKa’ (~ 7.4) observed for compound 1 are similar to those reported by Aigner et al. for a fluorescent probe for pH sensing in living cells based on PBI cores25. For that probe, the fluorescence lifetime response to pH was only attributed to the PET effect associated to the protonation of a secondary amine linked to the imide group through a two-carbon chain (the bulky groups on the periphery of the PBI core should suppress aggregation). The formation of large aggregates could limit the use of a dye in some sensing applications, such as those related to biological systems. Nevertheless, we have observed that the solutions of 1 are stable, an unlimited growth of the aggregates with



the time is not expected and, therefore, its use as a fluorescent probe in biological applications a priori cannot be ruled out. In this sense, fluorescent probes based on aggregation-induced emission effect for intracellular pH sensing have been recently reported48. The last part of this work is devoted to study the biocompatibility of 1 in living cells.

Figure 10. Fluorescence emission decays of a 1 µM solution of 1 at different pH values (λex = 565nm; λem = 590 nm).

4

Lifetime (ns)

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

Tris buffer SIB

3

2

1

3

4

5

6

7

8

9

10

11

pH


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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 11. Fluorescence lifetimes variation of 1 in Tris-HCl buffered solutions and in SIB solutions (concentration of the sample was 1 µM).

Table 2. Fluorescence lifetimes as a function of the pH and the concentration of the sample, [1] (λex = 565nm, λem = 590 nm) pH 5.2 [1] / µM

τm ± 2σ / ns

0.5

1

2.5

5

4.16 ± 0.09 4.20 ± 0.07 4.28 ± 0.05 4.30 ± 0.04

pH 7.4

pH 10.2

A0-0/A0-1

τm ± 2σ / ns

A0-0/A0-1

τm ± 2σ / ns

A0-0/A0-1

0.82

2.81 ± 0.08

0.62

0.57 ± 0.16

1.26

0.65

2.82 ± 0.08

0.52

0.63 ± 0.09

0.96

0.58

2.98 ± 0.07

0.46

0.54 ± 0.06

0.70

0.53

2.97 ± 0.14

0.45

0.43 ± 0.04

0.62

Biocompatibility. Cytotoxicity and cellular uptake are two key factors that must be considered for the potential use of a compound as a fluorescent probe for living cells. Cell viability was tested in C3H10T1/2 mesenchymal cells through the MTT assay. Cells were treated with different concentrations from 0.25 to 5.0 µM of compound 1 for 24h at 37°C. No significant effects on the cellular viability were found with the lower tested concentrations (0.25-1.0 µM). However, a decrease in cell viability was detected at higher concentrations. The cell morphology remained unchanged during the experimental incubation times within the range of tested concentrations (0.25-5.0 µM) (see Figure 12a). Uptake by C3H10T1/2 cells was detected after 1h of incubation in medium with 0.5 µM compound 1. Cellular uptake of 1 was examined by means of



confocal fluorescence imaging microscopy. As stated in the experimental section, C3H10T1/2 cells were treated with a 0.5 µM aqueous solution of 1 for 1 h. As shown in Figure 12, a strong fluorescence signal was detected in the cells, demonstrating cellular uptake of the PBI dye (see Figure 12b). The bright spots observed in the cells seem to indicate that the compound 1 is not uniformly distributed in the cytosol. Aigner et al. already suggested the selective localization of a related fluorescent probe (based on a PBI core) in endosomal compartments25. Our study offers new insights to understand the physical processes associated to the pH dependent fluorescence of PBI dyes and their use as fluorescent probes in sensing and biosensing applications.

(a)

100

*

*

80

% cell viability

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

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***

***

2.5

5

60 40 20 0 Control

0.25

0.5

0.75

1

[1] (µM) (b)


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Figure 12. (a) PBI cytotoxicity estimation by the MTT assay in C3H10T1/2 cells treated with 0.25 to 5.0 µM solution of 1 for 24h at 37ºC; (b) Confocal fluorescence image (i) and overlaid of bright field and confocal fluorescence image (ii) of living C3H10T1/2 cells at 37ºC following 1h of incubation with a 0.5 µM solution of 1. Image was obtained using λex = 488 nm and emission in the green channel.

CONCLUSIONS In this work, we have studied the photophysical properties of a functionalized PBI dye. Mainly, we have focused on the fluorescent properties of compound 1 (chart 1) as a function of the pH, its potential sensing applications and its effects on the viability of cells cultured in vitro. After a set of absorption spectroscopy experiments, it was observed that compound 1 shows a high tendency to form π-π aggregates in aqueous medium. Disaggregation only can be achieved by dilution, heating and acidification of the medium. The aggregation state of compound 1 changes with pH due to the protonation equilibria involving the amino groups of the side chains. At room temperature and working concentration, the presence of aggregates was readily observed in a sufficiently acidic medium ensuring the protonation of the amine groups (pH = 2.0). In basic medium, the significant changes reordering the absorption spectra suggested that the aggregates were more strongly coupled. The 1H-NMR spectra of 1 in



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D2O/TFA also demonstrated that aggregation state is dependent on concentration and temperature. The fluorescence excitation and emission spectra in aqueous solution showed the characteristic vibronic structure of non-aggregated PBI dyes since strongly coupled aggregates are efficiently quenched. As a consequence, the fluorescence intensity decreases with increasing pH and the resulting deprotonation of the amine groups. Besides aggregation, it was also demonstrated that the PET phenomenon could contribute to the fluorescence quenching in the uncharged state of 1. The dependence of the aggregation state with pH was also studied by means of DFT calculations. For this task, diverse molecular dimers and tetramers were optimized in different protonation states. According to our experimental observations, 1 remains aggregated when it is protonated. Intermolecular hydrogen bonds involving amine and carbonyl groups could help to maintain the stability of the molecular cluster. Sliding of a molecule on its neighbor and, therefore, an increased intermolecular distance were observed for clusters in a protonated state with respect to their deprotonated counterparts. Hence, the enhanced fluorescence emission recorded at acidic pH is consistent with the weaker coupling between the monomeric units in protonated clusters. In additional experiments, we observed that the fluorescence lifetime, τav, of 1 showed a sigmoidal dependence on pH with a linear response between pH 6 and 8, but sample concentration (within the range 0.5 – 5 µM) did not exert a significant influence. In presence of a synthetic intracellular buffer, the curve τav vs. pH exhibits a similar shape but the maximum τav values (for pH > 6.0) decreased from ~ 4.2 ns (in Tris-HCl buffer) to ~ 2.5 ns (in SIB buffer). Finally, the biocompatibility of compound 1 was assessed in C3H10T1/2 cells. No significant effect on cellular viability was observed at the concentration and time of treatment employed to evaluate the uptake in C3H10T1/2


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cells. The strong fluorescence signal observed in C3H10T1/2 cells confirmed the uptake of 1 following 1h of incubation. Similarly to what was previously reported for a related fluorescent probe based on PBI25, compound 1 did not distribute homogenously in the cytosol. The data presented here support the notion that an incubation of an hour with 0.5 µM concentration of compound 1 are adequate conditions to enable the use of compound 1 as a fluorescent probe in living cells. This study provides further insights on the mechanism associated to the fluorescence response with pH changes of this family of functionalized PBI dyes and its use as fluorescent probes in sensing applications.

SUPPORTING INFORMATION Supplementary Tables S1 and S2: relative energy and supramolecular structural parameters calculated for the molecular dimers in gas phase and for the molecular tetramers in aqueous solution, respectively. Supplementary Figures S1 – S6: NMR, FTIR and HRMS spectra of compound 1. Supplementary Figures S7 and S8: Absorption spectrum of compound 1 at basic pH and different temperatures; emission fluorescence anisotropy measured for aqueous solutions of 1 at different pH values.

ACKNOWLEDGEMENTS The authors would like to thank the `Consejería de Educación y Ciencia de la Junta de Comunidades de Castilla-La Mancha´ (Project PEII11-0279-8538) for financially supporting the research described in this article and the Ùniversidad de Castilla-La Mancha´ for additional support of the research group (grants GI20152958, GI20163548, GI20163441 and GI20163569). Likewise, authors are grateful for the project DIPUAB-



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

16-GARZONRUIZ financed jointly by the `Diputación de Albacete´ and the Ùniversidad de Castilla-La Mancha´. M. Moral thanks the E2TPCYTEMASANTANDER program for the financial support. M. Moral gratefully acknowledges Supercomputing Service of Castilla-La Mancha University for allocation of computational resources. P.J. Pacheco thanks the Ùniversidad de Castilla-La Mancha´ for his PhD fellowship.

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Würthner, F.; Saha-Möller, C. R.; Fimmel, B.; Ogi, S.; Leowanawat, P.; Schmidt, D. Perylene Bisimide Dye Assemblies as Archetype Functional Supramolecular Materials. Chem. Rev. 2016, 116, 962–1052.

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Wang, Y. J.; Li, Z.; Tong, J.; Shen, X. Y.; Qin, A.; Sun, J. Z.; Tang, B. Z. The Fluorescence Properties and Aggregation Behavior of Tetraphenylethene– perylenebisimide Dyads. J. Mater. Chem. C 2015, 3, 3559–3568.

(3)

Aigner, D.; Dmitriev, R. I.; Borisov, S. M.; Papkovsky, D. B.; Klimant, I. pHSensitive Perylene Bisimide Probes for Live Cell Fluorescence Lifetime Imaging. J. Mater. Chem. B 2014, 2, 6792–6801.

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Aigner, D.; Borisov, S. M.; Petritsch, P.; Klimant, I. Novel near Infra-Red Fluorescent pH Sensors Based on 1-Aminoperylene Bisimides Covalently Grafted onto Poly(acryloylmorpholine). Chem. Commun. 2013, 49, 2139.

(5)

Georgiev, N. I.; Sakr, A. R.; Bojinov, V. B. Design and Synthesis of Novel Fluorescence Sensing Perylene Diimides Based on Photoinduced Electron Transfer. Dye. Pigment. 2011, 91, 332–339.

(6)

Montalti, M.; Battistelli, G.; Cantelli, A.; Genovese, D. Photo-Tunable Multicolour Fluorescence Imaging Based on Self-Assembled Fluorogenic


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