pH-Controlled Self-Assembly of X-Shaped Conjugated Molecules: The

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C: Physical Processes in Nanomaterials and Nanostructures

pH-Controlled Self-Assembly of X-Shaped Conjugated Molecules. The Case of 1,2,4,5-Tetrastyrylbenzene Rocio Dominguez, Juan Tolosa, Mónica Moral, Ivan Bravo, Jesus Canales-Vazquez, Julián Rodrígue- López, Andrés Garzón-Ruiz, and Joaquin C. Garcia-Martinez J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.8b05024 • Publication Date (Web): 01 Aug 2018 Downloaded from http://pubs.acs.org on August 16, 2018

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The Journal of Physical Chemistry

pH-Controlled Self-Assembly of X-Shaped Conjugated Molecules. The Case of 1,2,4,5-Tetrastyrylbenzene Rocío Domínguez,a,b Juan Tolosa,a,b Mónica Moral,d Iván Bravo,c Jesús CanalesVázquez,d Julián Rodríguez-López,e Andrés Garzón-Ruiz*c and Joaquín C. GarcíaMartínez*a,b a

Department of Inorganic, Organic Chemistry and Biochemistry, Faculty of Pharmacy, University of Castilla-La Mancha, C/ José María Sánchez Ibañez s/n, 02008, Albacete, Spain. E-mail: [email protected] b Regional Center for Biomedical Research (CRIB), C/ Almansa s/n, 02008, Albacete, Spain. c Department of Physical Chemistry, Faculty of Pharmacy, University of Castilla-La Mancha, C/ José María Sánchez Ibañez s/n, 02008, Albacete, Spain. e-mail: [email protected] d Renewable Energy Research Institute, University of Castilla-La Mancha, Paseo de la Investigación 1, 02071, Albacete, Spain. e Department of Inorganic, Organic Chemistry and Biochemistry, Faculty of Chemistry and Technological Chemistry. University of Castilla-La Mancha. Adva. Camilo Jose Cela, 13. 13071, Ciudad Real (Spain).

Abstract The control of the self-assembly of organic material compounds has a significant impact on different technological, biomedical and sensing applications. In this work we have studied the influence of the nature of the side chain on the one-dimensional selfassembly of X-shaped conjugated molecules based on 1,2,4,5-tetrastyrylbenzene cores. This phenomenon was firstly studied in solution by means of different spectroscopic techniques and the experimental observations were compared to the behavior of a homologous compound with Y-shape, based on a 1,3,5-tristyrylbenzene core. The aggregation state of these compounds was controlled by pH and temperature of medium. Density Functional Theory calculations allowed to better understand protonation equilibria related to aggregation processes. Finally, the supramolecular 1 ACS Paragon Plus Environment

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ordering of the set of X-shaped molecules studied here in solid phase was analyzed through transmission electron microscopy.

INTRODUCTION Controlling the self-assembly of π-conjugated organic compounds is a challenging task with significant impact on different scientific disciplines.1,2,3,4 For instance, in the case of technological applications, the supramolecular organization of the active material has a dramatic effect on the semiconducting behavior and photophysical properties of electronic devices.2,3,4,5,6,7,8 Fascinating biomedical applications have also been proposed for self-assembled nanomaterials. An important number of optic, magnetic and electrochemical sensors based on large supramolecular aggregates have been reported for diagnostics and sensing applications.2,9,10 Sensors based on small-size organic molecules can also self-assemble in different experimental conditions modifying their performance.11,12 In addition, molecular self-organization can lead to soft materials employed in gene and drug delivery such as gels, liposomes, micelles, dendrimers, virus-like structures, polymersomes and dendrimersomes.2,10,13 Furthermore, in synthesis, catalysis, separation and purification are used self-organized molecular systems.2,14,15 In previous works, we have studied the self-assembly processes of the Y-shaped molecules based on a core of 1,3,5-tristyrylbenzene with different side chains.7,11 In general, discotic π-conjugated molecules tend to form columns which, in turn, organize themselves in 2D-lattices.3,4,16 Transmission electron microscopy (TEM) have shown how 1,3,5-tristyrylbenzene, and other discotic π-conjugated molecules as hexa-perihexabenzocoronene, form ordered structures in the solid state, in which lattice fringes that reveal the intracolumnar periodicity corresponding to the π-stacking of the discs are

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observed.4,7,17 Nevertheless, the type of aggregate depend on the nature and length of the side chains in addition to the shape of the π-conjugated core. The supramolecular ordering is controlled by the balance between different noncovalent interactions, i.e. π−π interactions between conjugated cores, as well as hydrogen bonds and van der Waals forces mainly associated to the side chains.3,4,18,19 Thus, the nature and length of these chains have a strong influence on the tilt angle and tilt direction of the columnar aggregates and determine the type of columnar mesophase. The role of the side chains in 2D assembly of 1,3,5-tristyrylbenzene and other discotic π-conjugated molecules has recently been investigated by several research groups using scanning tunneling microscopy (STM) on molecules deposited on metallic and graphite surfaces.20,21,22,23 Our goal here is to extend the study on self-assembly to a larger π-conjugated core with X-shape, i.e. 1,2,4,5-tetrastyrylbenzene, and different side chains, and obtain more general conclusions on the factors that control the supramolecular organization of poly(styryl)benzenes. With this aim in mind, three novel compounds with a same Xshaped core and different side chains have been synthetized. The side chains have the generic formula -CH2-NH2-(CH2)2-R, being R a hydroxyl group, a primary amine or a tertiary amine (see compounds 2a, 2b and 2c in Scheme 1). Note that compounds 1 and 2a share the same side chains but have different cores, i.e. Y-shaped core vs. X-shaped core. Recently, we have reported a study on the effect of the aggregation state ‒ controlled through the pH of medium‒ on the photophysical properties of 1.11 This compound will be used as reference in our later discussion. This study was conducted in three stages, i.e. (i) free molecule in solution, where spectroscopic measurements were carried out to analyze the differences of photophysical properties of Y-shaped and Xshaped molecules based on styrylbenzene; (ii) aggregated molecule in solution, which was studied through different steady-state and time-resolved spectroscopic experiments;



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and (iii) solid state, where the supramolecular ordering in the solid was investigated employing TEM. Aggregation processes are generally controlled through different experimental conditions such sample concentration, polarity of the solvent, pH and temperature,

among

others.20,24,25

Here,

the

aggregation

state

of

1,2,4,5-

tetrastyrylbenzene derivatives in solution was varied as a function of pH and temperature. Density Functional Theory (DFT) calculations were also employed to shed light on the protonation equilibria that take place in the amines of the side chains.

Scheme 1. Molecular formula of the studied compounds.

MATERIAL AND METHODS Synthesis. Compound 1 was obtained as reported previously.13 The synthesis of 2a-c starts from a tetrastyrylbenzene tretraaldehyde core26 that provides the π-conjugated building block with optical absorption and emission properties. Reaction with an excess of the three commercially available amines led to the tetraimines, which were reduced in situ with sodium borohydride to yield the desired compounds 2a-c (see Supporting Information for more details). Spectroscopic Measurements. Sample concentrations of 10 and 1 µM were employed for UV-Vis absorption spectroscopy experiments and fluorescence assays, respectively, 4 ACS Paragon Plus Environment

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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, dimethyl sulfoxide (DMSO) solutions of 2b and 2c were also titrated with trifluoroacetic acid (TFA) to assess whether the fluorescence signal was also quenched by the photoinduced electron transfer (PET) effect. Quartz cuvettes (Hellma Analytics) of 10 mm were employed for all the absorption and emission measurements in liquid samples. UV-Vis absorption spectra were acquired in a V-650 (Jasco) spectrophotometer and with a scan rate of 600 nm min‒1. A Peltier accessory was employed to control the temperature of the spectrophotometer measuring cell. FTIR measurements were carried out at room temperature using a 640-IR (Varian) spectrophotometer equipped with an attenuated total reflection (ATR) accessory. 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 temperaturedependence experiments). For SSF spectra, a 450 W Xe lamp was used, and the light source and the excitation and emission slits were both fixed at 3 nm, except where otherwise expressly indicated. The step and dwell time were 1 nm and 0.1 s, respectively. For TRF experiments, an EPLED360 sub-nanosecond pulsed light emitting diode (Edinburgh Photonics) was employed as light source at 368 nm, and the fluorescence decay profiles were collected at 460 nm. The fluorescence intensity



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decays, I(t), were fitted by using an iterative least square fit method to the following multiexponential function: = ∑ exp −/

(1)

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

=

∑

∑

(2)

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 320 nm, 10 nm and 0.35 nm, 1 nm and 0.5 s, respectively. Quantum yield calculations were carried out using the F980 Software of Edinburgh Instruments. Transmission Electron Microscopy (TEM). TEM studies were performed using a 2100 JEM (JEOL) electron microscope operating at 200 kV. Specimens for TEM observation were prepared by depositing aqueous solutions of the samples (0.1 µM) onto a holey carbon grid (EMS). Specimen observation was carried out under low-dose conditions to minimize beam damage. Computational Details. A theoretical study was achieved to understand in more detail protonation equilibria of compound 2c, which shows the more complex behavior with the pH. All the calculations were performed with the Gaussian09 (revision D.01) suite of programs27 and the open-source software Avogadro (revision 1.2.0).28 An initial random conformational search was carried out with Molecular Mechanics employing the force field MMFF9429,30,31,

32,33

in Avogadro.28 The ten lowest energy conformers

were selected for compound 2c and optimized at the B3LYP/3-21G* level of theory.34,35 6 ACS Paragon Plus Environment

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Later, the lowest energy conformer at the B3LYP/3-21G* level was reoptimized with a larger basis set, B3LYP/6-31G*, and in aqueous solution using the Polarizable Continuum Model (PCM) methodology.36,37 Finaly, two partially protonated species (with four protons) and a fully protonated one (with eight protons) were modeled in aqueous solution too. The two partially protonated species correspond to the states in which all the inner (secondary) amines are protonated or all the outer (tertiary) amines are protonated. ∆Gr0 of the protonation equilibria was calculated as ∆ = ∑ Δ"

(3)

where ∆Gf0 is the formation free energy of the reactants and products in aqueous solution and υ is the stoichiometric factor (negative for the reactants and positive for the products). As known, computational methods poorly reproduce the solvation energies of the proton. A free energy of -270.28 kcal mol‒1 in aqueous solution was used for it following the recommendation of Camaioni and Schwerdtfeger.38 The calculation of the infrared spectra was also carried out at the B3LYP/6-31G* level of theory using a scaling factor of 0.9613, recommended in the literature for this combination of method and basis set.39

RESULTS AND DISCUSSION Spectroscopic Characterization. The UV-Vis absorption spectra of compounds 2a, 2b and 2c are shown in Figure 1(a), along with spectrum of 1 for comparative purposes. These spectra were recorded in 0.1 M HCl aqueous solutions, after 1 h of sonication, to break molecular aggregates. As expected, 2b and 2c exhibit similar absorption maximum wavelengths (λabmax = 327 and 329 nm, respectively), which are consistent with the values of 334 and 335 nm measured in methanol for these compounds (Figure S3 and S4, Supporting Information). This suggest that molecules are non-aggregated in



HCl solution. Additionally, similar values are observed in literature for other molecules with 1,2,4,5-tetrastyrylbenzene core in toluene and dichloromethane solutions.40,41 These absorbance wavelengths are about 10 nm red-shifted with respect to λabmax reported for 111 (λabmax = 317 nm) because the more effective conjugation of the para and orto arrangement styrylbenzene derivatives. On the contrary, the absorption of 2a shows a monotonically increasing absorbance toward higher energy because the scattering, along with a significant red-shift of the main band (λabmax = 350 nm) with respect to its counterparts, 2b and 2c. The absorption spectrum of styrylbenzene derivatives mainly depends on the conjugated core,7,40,42 but the side chains can play a key role in the control of the aggregation state of these compounds and, in turn, produce spectral shifts.1,2,3,4,11 We assume that 2a is aggregated despite of the drastic experimental conditions employed. In very similar way, fluorescence spectra of compounds 2b and 2c (Figure 1(b)) are consistent with previously reported for similar cores and maximum emission wavelengths (λemmax = 455 and 451 nm, respectively), are red-shifted compared with compound 1. (λemmax = 413 nm). Once again, aggregation of compound 2a, even in very acid media, yields a very broad and far red-shifted emission band (λemmax = 564 nm).


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Figure 1. Different spectra recorded for the studied compounds in 0.1 M HCl aqueous solution and 296 K. (a) Absorption spectra (sample concentrations were 10 µM); (b) normalized fluorescence emission spectra (sample concentrations were 1 µM).



Table 1. Absorption, Excitation and Emission Maximum Wavelengths (λabmax, λexmax and λemmax) for Compounds 1, 2a, 2b and 2c in 0.1 M HCl at 296 K Compound λabmax (nm) λexmax (nm)a λemmax (nm)b c c 317 315 413c 1 350, [~455(sh)]d 364 564 2a d [~295(sh)], 327 [~304(sh)], 330 455 2b d [~295(sh)], 329 329 451 2c a max Excitation spectra were collected at λem b Emission spectra were collected at λabmax c Data from reference 11. d Two peaks can be distinguished in the absorption band. (sh) corresponds to a shoulder.

Study of the Aggregates in Aqueous Solution. As previously studied by our group, styrylbenzene compounds have a strong tendency to form aggregated species in different experimental conditions.7,11 In this work, we have analyzed the effect of the pH and temperature on the aggregation state of the set of studied 1,2,4,5tetrastyrylbenzene derivatives. These compounds have side chains with different amine groups and the protonation state of these groups may play a role in self-assembly processes. In a previous work, we concluded that the side chains of compound 1 favors the aggregation due to the formation of hydrogen bonds between amines of different molecules.11 Compound 1 is disaggregated in low pH aqueous solutions and forms Haggregates upon pH increase. On the contrary, spectra obtained for compound 2a in different experimental conditions show that it remains aggregated even though at low pH values and high temperatures. The significant decrease of the absorbance of 2a at higher pH values (see spectrum (a) in Figure 2) indicates that the aggregation process progresses owing to the deprotonation of the amines. In an opposite direction, Figure 2(b) shows how the absorption maximum of 2a enhances and shifts ~10 nm to the blue upon heating but is still far from the absorption maximum of 2b and 2c. Comparing the aggregation behavior of compounds 1 and 2a, it is possible to conclude that 1,2,4,510 ACS Paragon Plus Environment

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tetrastyrylbenzene derivatives form stronger aggregates than 1,3,5-tristyrylbenzene derivatives, possibly due to the larger size of the π-conjugated system and higher number of side chains which can establish intermolecular H-bonds between amine groups. For compound 2b, the band with the absorption maximum centered at 328 nm enhances with the pH value up to pH ~ 6 but drastically decreases for pH > 6 (see Figure 2(c)). The shape of the band changes from acidic to basic solution, i.e. a slight blue shift (∆λ ~ -5 nm for pH = 7.9) and the apparition of a long band tail in the red are observed in basic medium. An isosbestic point is clearly observed at 395 nm indicating the equilibrium between two species. Figure 2(d) suggests the formation of aggregated species seems to be stable up to 70ºC. Finally, a dramatic drop in the absorption intensity was observed for compound 2c when the pH increases but the formation of the aggregate does not produce significant changes in the shape of the spectrum (see Figures 2 (e-f)). The lack of spectral shifts in 2c could be related to a weaker intermolecular coupling in the aggregated state due to the higher steric hindrances of its side chains. Aggregation of 2b and 2c is reversible, although we have observed that these processes are generally faster than disaggregation.



Figure 2. Evolution of the absorption spectra as a function of pH and temperature. Spectra (a,b) corresponds to compound 2a, spectra (c,d) corresponds to compound 2b and spectra (e,f) corresponds to compound 2c. Sample concentrations were 20, 50 and 100 µM for compounds 2a, 2b and 2c, respectively. Experiments as a function of the temperature were carried out in a 0.1 M HCl solution for 2a and at two different pH values for 2b and 2c.

Additional fluorescence emission spectroscopy experiments were carried out to shed light on the aggregation processes. For simplicity, we shall start the discussion for compound 2b that only has one type of amine in the side chain. Figure 3(b) shows how the emission intensity decreases with the pH for values beyond 6, in parallel to the results of absorption spectroscopy. This compound exhibits the highest fluorescence quantum yield (ΦF) of the series of 1,2,4,5-tetrastyrylbenzene derivatives in acidic 12 ACS Paragon Plus Environment

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solution, i.e. ΦF = 21.7% for pH = 4.0 and slightly lower for pH = 7.0 (see Table 2). However, in accordance with the previous spectroscopy experiments, ΦF drops to 4.9 % for pH = 11.0 where we assume that the molecule is aggregated. Figure 4 points out the evolution of fluorescence emission decays as a function of pH. The curve of fluorescence lifetimes, τF, vs. pH shows a classical S-shape with a pKa = 9.1 which is consistent with the pKa reported for benzylethylamine (9.68).

43

In addition to the

aggregation-quenched emission mechanism, we also investigated the possible role of PET processes in the quenching of the electronic excited state of these molecules. Figure 5 shows how the fluorescence intensity of 2b in DMSO solution enhances upon titration with TFA up to a concentration ration of [TFA]/[2b] = 4 and then it stays constant. Assuming that 2b is free in solution (DMSO is a good solvent for 2b), the increase of the emission intensity seems to be associated to the protonation of the four amines and the block of PET process. For compound 2c, pH-dependence of fluorescence emission exhibits a more complex behavior since there are two different amino groups in the side chains. In the experiments (see Figures 3(c) and 4, and Table 2), two different patterns can be differentiated: (i) fluorescence intensity and lifetime increase with the pH within the range pH = 4 – 8; (ii) fluorescence intensity and lifetime decreases with the pH within the range pH = 8 – 10 and remain fairly constant for pH > 10. Table 2 shows how quantum yields also follow the same behavior. Considering the pKa values reported for benzylethylamine (9.68) and dimethylethylamine (10.16), the first protonation —going from basic to acid conditions— should occur in the tertiary (outer) amines and the second one in the secondary (inner) amines.43,44 Nevertheless, the closeness of both pKa values does not allow definitive conclusions on the protonation equilibria of 2c and a thorough analysis is needed. Firstly, the fluorescence intensity profile observed in



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Figure 5 for 2c, in DMSO solutions, is different to the previous one recorded for 2b. This fact seems to indicate that the first protonation should occur in a different type of amine (the outer amines) but the kind of solvent could also play a role in the protonation equilibria. Thus, protonation equilibria of 2c were also studied by means of DFT calculations. Figure 6 shows the optimized geometry of compound 2c in aqueous solution and different protonation states, i.e. deprotonated state (2c), partially protonated state with four protons (2ci and 2co corresponds to the protonation of the inner and outer amines, respectively) and fully protonation state with eight protons (2cf). Thus, the first protonation equilibria considered can be written as follow

#$ + 4H ( ⇄ #$*

(4)

#$ + 4H ( ⇄ #$+

(5)

The lowest reaction free energy was calculated for eq. (5) (∆G2Ci = -40.0 kcal mol-1 and ∆G2Co = -47.7 kcal mol-1) indicating that the most thermodynamically stable product derives from the protonation of the outer amines. In this sense, it can be observed how the state 2co is additionally stabilized by H-bonds between the protons and the inner amines (H···N distance of these interactions is within 2.00 and 2.04 Å, and is shorter than the corresponding sum of van der Waals radii of both atoms).45 This could also explain why partially protonated form of 2c is more fluorescent than the neutral species. That is, the first protonation reaction avoids the quenching effect associated to the aggregation and, at the same time, could partially block the PET effect due to the hydrogen bonds established by the inner amines. Regarding the fully protonated form of 2c, Figure 6(c) shows how compound 2c adopts a more extended form in this state than in partially protonated or neutral states. This could be related to the decrease of the fluorescence intensity (and quantum yield) at acid pH values. In the `open configuration´ of the fully protonated state, side chains provide less hydrophobic


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environment to the conjugated core and less protection against the fluorescence quenching effect of solvent molecules, oxygen and impurities. For instance, oxygen also accelerates the efficiency of the process of E-to-Z isomerization in 1,3,5tristyrylbenzene derivatives.42 It is known that 1,3,5-tristyrylbenzene derivatives with dendritic side chains generally have higher quantum yields than compounds with shorter chains.11,42 As expected, no significant changes were observed in the shape of the fluorescence emission spectrum of 2a within the range of pH studied. ΦF and τF also remain fairly constant with a slight drop at high pH values. This compound seems to stay in a similar aggregation state at the different experimental conditions. Disaggregation of 2a was not achieved despite the drastic experimental conditions tested (0.1 M HCl solution and sonication during 2 h). The molecule 2a has eight primary and secondary amines and the formation of intermolecular H-bonds can lead to strong aggregates difficult to break. In contrast, steric hindrances of the tertiary amines of compound 2c seems to be a key factor that weakens intermolecular interactions and favors the solubility.



Figure 3. Fluorescence emission spectra of compounds 2a, 2b and 2c as a function of the pH of the medium. Sample concentrations were 1 µM.


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Figure 4. Average emission lifetimes (τF) measured for compound 2a, 2b and 2c at different pH values.

Figure 5. Evolution of fluorescence emission intensity (F) of compounds 2b and 2c upon titration with TFA in DMSO solution. F0 corresponds to the fluorescence intensity recorded before the addition of TFA. (λex = 330 nm; λem = 455 nm; concentration of the sample was 1 µM).



Figure 6. Molecular structure calculated for different protonation forms of compound 2c at the B3LYP/631G* level of theory. Picture (a) corresponds to 2ci, the partially protonated state with four protons in the inner amines; while picture (b) shows the molecular structure of 2co, with the outer amines protonated. Picture (c) shows the superimposed structures of the neutral state of 2c (in red color) and its fully protonated form (in green color).


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Table 2. Fluorescence Quantum Yields (ΦF) Measured for Compounds 2a, 2b and 2a at Different pH Values pH 4.0 7.0 11.0

2a 3.5 3.5 2.6

ΦF (%) 2b 21.7 20.6 4.9

2c 9.7 19.8 3.5

Solid State Experiments. TEM micrographs of 1,2,4,5-tetrastyrylbenzene derivatives in the solid state are shown in Figure 7. These compounds give rise to nanometric ordered structures inside larger amorphous aggregates and fibers. Similar ordered structures showing narrow lattice fringes were already observed for compound 1 (with a periodicity of 0.34 ± 0.10 nm) and for other discotic π-conjugated molecules as hexaperi-hexabenzocoronene (with a periodicity of 0.49 nm).11,17 These narrow lattice fringes were attributed to the intracolumnar periodicity corresponding to the π-stacking of the discs.11,17 In the case of hexa-peri-hexabenzocoronene, wider lattice fringes with periodicities of 2.48 nm were also observed in TEM micrographs and were assigned to the intercolumnar distance.17 In addition to the π-conjugated core, the nature of side chains also plays a key role in the supramolecular ordering of discotic conjugated molecules. For instance, a significantly larger lattice-fringe spacing (0.88−0.92 nm) was found for another 1,3,5-tristyrylbenzene derivative related to 1, but having hexyloxy side chains.7 The establishment of intermolecular hydrogen bonds between the amines of the peripheral chains of 1 can explain the shortening of the lattice-fringe spacing. The existence of hydrogen bonds between amine groups, in the solid state, was confirmed for the studied compounds by means of FTIR spectroscopy (Figure S11). The bands corresponding to H–N stretching modes are significantly broad indicating the formation of H-bonds. Additionally, Figure S11c shows a comparison between the broad band recorded for compound 2c in the solid state (3500 - 3100 cm-1) and the corresponding



normal mode frequency calculated for this molecule in the isolated state approximation (3340 cm-1). The microscopy experiments carried out in this work allow going in depth into the effect of the core and the side chains on the internal order of styrylbenzenes in the solid state. Thus, in a first stage, the comparison of the TEM images obtained for compounds 1 and 2a is highly relevant as they exhibit the same type of side chains but different core. For compound 2a, nanodomains, with a diameter of 3 – 10 nm, were found inside larger amorphous aggregates (see Figure 7). Such nanometric structures show high degree of internal order with a periodicity of 0.20 ± 0.02 nm between lattice fringes. The larger size of the conjugated core of 2a with respect to 1 favors π-stacking interactions and seems to approximate the molecules in the supramolecular structure. The lattice-fringe spacing found for 2a is also significantly shorter than the spacing of 2b and 2c, showing that this combination of core and side chains maximizes the intermolecular interactions (π-stacking and H-bonds) and it is consistent with the great insolubility of compound 2a. These interactions seem to be especially favored through the stacking direction, in detriment of coplanar interactions since, curiously, compound 2a forms smaller periodic structures than the rest of compounds. More extended periodic structures (10 – 50 nm), with trapezoidal form, were found for 2b which show lattice fringes with a periodicity comparable to compounds 1 and 2c (0.32 ± 0.03 nm and 0.34 ± 0.04 nm for 2b and 2c, respectively). Interestingly, some crossed lattice fringes can be observed for 2b indicating that the nanometric aggregates could be formed by the accumulation of layers with molecules oriented in different directions. The lattice fringes observed for 2b are straight in contrast to those found for 2c, which are curved and define long fibers of tenths of nanometers. The tertiary amines of 2c could provoke intermolecular steric hindrances and the folding of the periodic structure. In contrast, the combination of 1,2,4,5-tetrastyrylbenzene core


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and -CH2NH(CH2)2OH side chains (compound 2b) favors the establishment of cofacial (intracolumn) and coplanar (intercolumn) interactions, which extend straight periodic structures tenths of nanometers.

Figure 7. TEM images of 2a (a), 2b (b) and 2c (c) from an aqueous solution.



CONCLUSIONS In this work, we have studied the photophysical properties of X-shaped molecules based on styrylbenzene and compared them to those reported for Y-shaped molecules. These molecules tend to aggregate in basic aqueous solution while the protonation of the amine groups of their side chains in acidic medium generally avoids aggregation. The nature of the side chains along with the π-conjugated core control the aggregation process. For compound 2b, the S-shape found for the titration curve of fluorescence lifetime as a function of pH (pKa = 9.1) was attributed to the protonation equilibrium of the single type of amine of the side chains. Additional experiments in an organic solvent solution titrated with TFA demonstrated that the fluorescence of 2b in neutral state is not only quenched by aggregation but PET effect can play a role. Contrary to compound 2b, two different behaviors were observed in the corresponding titration curve of 2c. Firstly, within the range pH = 4 – 8, fluorescence intensity and lifetime increase with the pH. In view of the results obtained in the DFT study, the first deprotonation equilibrium seems to occur in the inner (secondary) amines and the conformational changes in the side chains can provide a more hydrophobic environment in the core and protection against the fluorescence quenching. Nevertheless, the inner amines can stablish H-bonds with the protonated outer amines giving rise to a side chain cyclization. This fact could partially block the PET effect and avoid a strong decrease of the fluorescence intensity. Afterwards, fluorescence intensity and lifetime decrease as consequence of the deprotonation of the outer amines. In general, the spectral shifts observed for compound 2b were more significant than for 2c evidencing that the terminal tertiary amines could produce steric hindrances to assembly and lead to weakly coupled aggregates. Concerning 2a, we found that the complete disaggregation of this compound is not produced in acidic aqueous solution in contrast to compound 1. No


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significant spectral shifts were observed for 2a within the range of pH studied, and both ΦF and τF remain fairly constant. Hence, we can conclude that 1,2,4,5tetrastyrylbenzene derivatives form stronger aggregates than 1,3,5-tristyrylbenzene derivatives due to the larger size of the π-conjugated system and higher number of side chains which can establish H-bonds between amine groups. The nature of the side chain also controls the supramolecular order in the solid state. TEM micrographs show nanometric ordered structures inside lager amorphous aggregates and fibers. These nanometric structures show high degree of internal order with periodicities ranging from 0.20 to 0.34 nm. The lattice-fringe spacing found for 2a is significantly shorter than the spacing of 1, 2b and 2c, indicating that this combination of core and side chains maximizes the intermolecular interactions. The tertiary amines of 2c could provoke intermolecular steric hindrances and the folding of the periodic structure, on the contrary, lattice fringes observed for 2a and 2b are straight.

SUPPORTING INFORMATION Synthetic procedures for the synthesis of compounds 2a, 2b, and 2c. 1H NMR and 13C NMR spectra. UV-Vis absorption spectra in MeOH. Fluorescence emission decays and fit result at different pHs.

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

Notes



The authors declare no competing financial interest

ACKNOWLEDGMENT The authors would like to thank the Ministerio de Economía y Competitividad (Spain) (project CTQ2017-84561-P) for supporting the research described in this article and the “Universidad de Castilla-La Mancha” for additional support of the research group (grants GI20163441 and GI20173955). RD thanks Junta de Comunidades de Castilla-La Mancha, Fondo Social Europeo (FSE) and Iniciativa de Empleo Juvenil (EIJ) for the postdoctoral grant. MM gratefully acknowledge Supercomputing Service of Castilla-La Mancha University for allocation of computational resources.

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