Design of Novel Pyrene-Bodipy Dyads: Synthesis, Characterization, Optical Properties, and FRET Studies

A new series of dendronized bodipys containing pyrene units was synthesized and characterized. Their optical and photophysical properties were determined by absorption and fluorescence spectroscopy. This series includes three different compounds. The first one has an anisole group linked to the bodipy unit, which was used as the reference compound. In the second, the bodipy core is linked to a zero generation dendron with one pyrene unit. The third compound contains a first generation Fréchet-type dendron bearing two pyrene units. In this work, the combination pyrene-bodipy was selected as the donor-acceptor pair for this fluorescence resonance energy transfer (FRET) study. Doubtless, these two chromophores exhibit high quantum yields, high extinction coefficients, and both their excitation and emission wavelengths are located in the visible region. This report presents a FRET study of a novel series of pyrene-bodipy dendritic molecules bearing flexible spacers. We demonstrated via spectroscopic studies that FRET phenomena occur in these dyads.


Introduction
The development of new photoactive compounds bearing donor-acceptor groups has very relevant in materials science [1][2][3]. In order to understand the energy transfer process that occurs in natural chlorins, many chemists have developed and designed more efficient materials for light energy conversion [3][4][5]. The fluorescence resonance energy transfer (FRET) phenomenon is a non-radiative process that includes the energy transfer between two molecules or two different groups in a molecule [6][7][8][9][10][11][12]. One of the molecules or groups is in the ground state (acceptor), whereas the other is in the first excited state (donor). The FRET phenomenon occurs via a Förster mechanism when the emission spectra of the donor group overlap, partially or totally, with the absorption spectra of the acceptor.
Bodipy (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) dyes have attracted the attention of many research groups due to its special photophysical properties. They present a strong ultraviolet (UV) absorption, relatively sharp fluorescence peaks with high quantum yields, and very good photostability [13][14][15][16][17]. Furthermore, they are relatively insensitive to the polarity and pH of the environment, and are reasonably stable to physiological conditions, showing high solubility in many organic solvents [15,18]. The main aspect that make bodipy dyes photophysically suitable is the tunable emission/absorption by functionalization [19,20].
We selected pyrene as the donor and bodipy as the acceptor chromophores in order to carry out a FRET study, since the emission spectrum of pyrene significantly overlaps the absorption spectrum of bodipy. A few reports are presented in the literature using this pair of chromophores, where they are directly linked or are attached via a short rigid spacer [19,[30][31][32][33]. The idea of this work was to connect both moieties through alkylic flexible chains and Fréchet type dendrons, allowing these chromophores to encounter one another, and for the orientation of their dipole moments to occur at a distance not longer than 10 nm, which are the conditions for the FRET phenomenon to occur [34]. Moreover the use of a Fréchet type dendron allowed us to obtain a molecular antenna, where more than one donor group can be attached to only one acceptor.

Synthesis of the Pyrene-Bodipy Compounds
The synthesis of pyrene-labeled dendrons 3 and 6 was carried out following the classical convergent method shown in Scheme 1. The synthetic route of the bromide precursors 2 and 5 was previously reported by our research group [24]. A nucleophilic substitution of compounds 2 or 5 with p-hydroxybenzaldehyde was carried out using K 2 CO 3 as a base and 18-crown-6 as a catalyst. After purification by column chromatography, the desired compounds 3 and 6 were obtained as white viscous solids with yields of about 80%. Proton nuclear magnetic resonance ( 1 H NMR), carbon NMR ( 13 C NMR), and electrospray ionization (ESI) mass spectroscopy confirmed the chemical structures of compounds 3 and 6. Notably, pyrene possesses outstanding photophysical properties, such as high quantum yield, long lifetime, and ease of formation of excimers [21][22][23][24][25][26][27]. For these reasons, our group focused our attention on the study of dendritic molecules bearing pyrene as the donor group and different acceptor groups, such as porphyrin, fullerene, and bispyridinium organometallic complexes, among others [24,28,29].
We selected pyrene as the donor and bodipy as the acceptor chromophores in order to carry out a FRET study, since the emission spectrum of pyrene significantly overlaps the absorption spectrum of bodipy. A few reports are presented in the literature using this pair of chromophores, where they are directly linked or are attached via a short rigid spacer [19,[30][31][32][33]. The idea of this work was to connect both moieties through alkylic flexible chains and Fréchet type dendrons, allowing these chromophores to encounter one another, and for the orientation of their dipole moments to occur at a distance not longer than 10 nm, which are the conditions for the FRET phenomenon to occur [34]. Moreover the use of a Fréchet type dendron allowed us to obtain a molecular antenna, where more than one donor group can be attached to only one acceptor.

Synthesis of the Pyrene-Bodipy Compounds
The synthesis of pyrene-labeled dendrons 3 and 6 was carried out following the classical convergent method shown in Scheme 1. The synthetic route of the bromide precursors 2 and 5 was previously reported by our research group [24]. A nucleophilic substitution of compounds 2 or 5 with p-hydroxybenzaldehyde was carried out using K2CO3 as a base and 18-crown-6 as a catalyst. After purification by column chromatography, the desired compounds 3 and 6 were obtained as white viscous solids with yields of about 80%. Proton nuclear magnetic resonance ( 1 H NMR), carbon NMR ( 13 C NMR), and electrospray ionization (ESI) mass spectroscopy confirmed the chemical structures of compounds 3 and 6. Scheme 1. Synthetic route for the aldehydic compounds 3 and 6. Reaction conditions: K2CO3, 18-crown-6, dimethylformamide (DMF), 110 °C, and four hours (yield 86% for 3 and 79% for 6).
The aim of the second part of the synthesis was to obtain the final compounds bearing bodipy and pyrene units. In order to have a reference, compound 1 was synthesized according to a The aim of the second part of the synthesis was to obtain the final compounds bearing bodipy and pyrene units. In order to have a reference, compound 1 was synthesized according to a procedure previously reported in the literature [14]. Generally, the synthesis of bodipy compounds starts with a condensation between an aldehydic compound and two equivalents of pyrrole in the presence of trifluoroacetic acid (TFA) as catalyst [13,16,35]. In our case, compounds 3 and 6 were employed to generate the new series (Scheme 2). After four hours, an oxidation in presence of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) was performed, in order to oxidize position 8. Finally, the bodipy compounds were obtained by addition of NEt 3 as catalyst and BF 3 O(Et) 2 . After purification by column chromatography, compounds 1, 4, and 7 were obtained as red solids with a yield of about 50%. 1 H NMR, 13  procedure previously reported in the literature [14]. Generally, the synthesis of bodipy compounds starts with a condensation between an aldehydic compound and two equivalents of pyrrole in the presence of trifluoroacetic acid (TFA) as catalyst [13,16,35]. In our case, compounds 3 and 6 were employed to generate the new series (Scheme 2). After four hours, an oxidation in presence of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) was performed, in order to oxidize position 8. Finally, the bodipy compounds were obtained by addition of NEt3 as catalyst and BF3O(Et)2. After purification by column chromatography, compounds 1, 4, and 7 were obtained as red solids with a yield of about 50%. 1 H NMR, 13

Optical and Photophysical Properties of the Pyrene-Labeled Bodipy Compounds
The optical and photophysical properties of the bodipy analogue 1, pyrene-labeled aldehyde precursors 3 and 6, as well as those of the pyrene-labeled bodipy compounds 4 and 7, were evaluated in THF solution. The first step in our work was to analyze if the pyrene and bodipy chromophores were interacting in the ground state when they are not linked covalently to each other. Therefore, a solution compound 1 (1.2 × 10 −5 M) was prepared. Firstly, the absorption spectrum of compound 1 was recorded; it corresponded to an absorption value of 1 at 501 nm. Then, a solution of 1-pyrenebutanol (2.4 × 10 −5 M) was prepared using the solution of compound 1 as a solvent in order to maintain the concentration of 1 constant. One consecutive dilution of 1-pyrenebutanol (1.2 × 10 −5 M) was performed. As a result, the absorption spectra demonstrated a regular increase in the absorption band corresponding to the pyrene moiety at 344 nm, whereas the part of the spectra corresponding to the absorption of the bodipy reference compound at 501 nm remained unchanged ( Figure 1). Therefore, we concluded that there are no interactions between these two chromophores when they are not linked in a chemical structure.

Optical and Photophysical Properties of the Pyrene-Labeled Bodipy Compounds
The optical and photophysical properties of the bodipy analogue 1, pyrene-labeled aldehyde precursors 3 and 6, as well as those of the pyrene-labeled bodipy compounds 4 and 7, were evaluated in THF solution. The first step in our work was to analyze if the pyrene and bodipy chromophores were interacting in the ground state when they are not linked covalently to each other. Therefore, a solution compound 1 (1.2 × 10 −5 M) was prepared. Firstly, the absorption spectrum of compound 1 was recorded; it corresponded to an absorption value of 1 at 501 nm. Then, a solution of 1-pyrenebutanol (2.4 × 10 −5 M) was prepared using the solution of compound 1 as a solvent in order to maintain the concentration of 1 constant. One consecutive dilution of 1-pyrenebutanol (1.2 × 10 −5 M) was performed. As a result, the absorption spectra demonstrated a regular increase in the absorption band corresponding to the pyrene moiety at 344 nm, whereas the part of the spectra corresponding to the absorption of the bodipy reference compound at 501 nm remained unchanged (Figure 1). Therefore, we concluded that there are no interactions between these two chromophores when they are not linked in a chemical structure. Compound 1 presented the typical absorption band of a bodipy chromophore [31,36,37]. The molar absorption coefficient for the most intense band at 501 nm was calculated to be 87,000 M −1 cm −1 . Furthermore, an interesting band between 320 and 400 nm was also observed. This absorption band was due to n-π* of the free electron pair in the methoxy group [38]. We calculated the molar absorption coefficient, with a value of about 6400 M −1 cm −1 at 362 nm in THF solution.
The absorption spectra of compound 4 presented the typical pyrene transition band at 344 nm due to the S0 → S2 transition [23,39] and the bodipy band at 501 nm for the S0 → S1 transition [31,35,36,40] (Figure 2). The spectra showed that the previously characterized band (320-400 nm) was preserved. We calculated the molar absorption coefficient for the two most intense bands at 344 and 501 nm (Table 1) in THF solution. Compound 7 showed higher pyrene absorption in comparison with compound 4 due to the presence of two pyrene units (Figure 3). The absorption coefficients were calculated at the same wavelengths (Table 1). Compound 1 presented the typical absorption band of a bodipy chromophore [31,36,37]. The molar absorption coefficient for the most intense band at 501 nm was calculated to be 87,000 M −1 cm −1 . Furthermore, an interesting band between 320 and 400 nm was also observed. This absorption band was due to n-π* of the free electron pair in the methoxy group [38]. We calculated the molar absorption coefficient, with a value of about 6400 M −1 cm −1 at 362 nm in THF solution.
The absorption spectra of compound 4 presented the typical pyrene transition band at 344 nm due to the S 0 → S 2 transition [23,39] and the bodipy band at 501 nm for the S 0 → S 1 transition [31,35,36,40] ( Figure 2). The spectra showed that the previously characterized band (320-400 nm) was preserved. We calculated the molar absorption coefficient for the two most intense bands at 344 and 501 nm (Table 1) in THF solution. Compound 7 showed higher pyrene absorption in comparison with compound 4 due to the presence of two pyrene units (Figure 3). The absorption coefficients were calculated at the same wavelengths (Table 1).        [40,41]. c All the Φbodipy were calculated using fluorescein in NaOH 0.1 M solution as the reference (0.92) [42]. The fluorescence spectra of compounds 3, 4, 6, and 7 were measured in THF solution at room temperature with a concentration corresponding to an absorption of about 0.03 at excitation wavelength (344 nm). Compound 3 showed the typical monomer emission band of the pyrene moiety, whereas the emission spectra of compound 6 showed monomer as well as excimer emission, due to the presence of two pyrene units in the same molecule (Supporting Information Figures S2 and S3).
Compound 4 showed that the pyrene emission between 360 and 480 nm was almost totally quenched and presented a strong emission band corresponding to the bodipy unit at 509 nm ( Figure 2). This is due to the presence of a FRET phenomenon. The same behavior was observed in compound 7, whose emission spectrum showed the same characteristics even though there are two pyrenes units in the same molecule ( Figure 3).
The possibility that a FRET process occurs between the pyrene donor and the bodipy acceptor was evaluated through the determination of the Förster radius (R 0 ) for these pair of chromophores using the following equation: where k is the orientation factor (2/3), n is the refractive index of the solvent (THF 1.4050), φ is the fluorescence quantum yield of the 1-pyrenebuthanol (value), and J is the overlap integral of the two chromophores, calculated using the following equation [40]: where f D is the donor emission spectrum that was normalized to its area, ε A is the molar absorption coefficient of the acceptor, and λ is the wavelength. As a result, the R 0 value for this pair of chromophores in THF solution was calculated to be 2.208 nm. Considering the flexibility of the chemical structures, it is reasonable to estimate that, in solution, the two parts of the molecule can be brought closer together than the calculated Förster radius, and therefore the FRET phenomenon can easily occur.
The quantum yields of compounds 3, 4, 6, and 7 were calculated ( Table 1). All the experiments were carried out in triplicate. In the case of compounds 3 and 6, due to their sensibility to oxygen, degassed solutions were required. The emission of the bodipy compounds 4 and 7 was not significantly affected by oxygen.
The difference in the quantum yields of pyrene in the dendrons and pyrene in the final compounds was due to the FRET phenomenon, which totally quenched the emission of this chromophore. When the excitation was performed at 344 nm, the values of the quantum yields showed that the FRET phenomenon occurs for compounds 4 and 7. The bodipy part received all the energy from the pyrene transition S 2 → S 0 and subsequently emitted this energy at 510 nm. Particularly, in the case of compound 1, a lower quantum yield was found at this wavelength confirming that the FRET phenomenon occurred. The quantum yields, calculated at 475 nm, exhibited comparable values to the one reported in the literature for a similar bodipy [17]. Compounds 4 and 7 showed a high FRET efficiency. Definitively, this study showed that between bodipy and pyrene FRET optimally occurs.

General Notes
Boron trifluoride diethyl etherate (BF 3 O(C 2 H 5 ) 2 ) and 2,4-dimetylpyrrole were freshly distilled under reduced pressure before use. The solvents employed in the reaction were purified by distillation in the presence of drying reagents (metallic sodium for THF or CaH 2 for dichloromethane). All the synthetic procedures were carried out under an argon atmosphere. The purification of the products was performed by column chromatography in silica gel and the elution was checked by thin layer chromatography (TLC). All intermediates and final compounds were characterized by 1 H NMR and 13 C NMR spectroscopies and MALDI-TOF mass spectrometry. NMR spectra were carried out on a Bruker Avance 400 MHz, operating at 400 MHz and 100 MHz for 1 H and 13 C, respectively. Samples were dissolved in deuterated chloroform. MALDI-TOF mass spectra were recorded using dithranol as matrix on a Bruker Daltonics Flex Analysis. UV-Visible (UV-Vis) spectra were carried out on a Unicam UV300 spectrophotometer using quartz cells with a width of 1 cm and THF (HPLC grade) as the solvent. Fluorescence spectra were recorded on a Fluorolog 3 spectrophotometer from Horiba with a xenon lamp as light source. The slit width for excitation and emission were defined to 1 nm.

Conclusions
A new series of dyads bearing pyrene-bodipy units as the donor-acceptor pair was synthesized and characterized. We demonstrated that both chromophores do not interact in the ground state. The spectroscopic studies proved that a FRET phenomenon occurs in dyads 4 and 7. FRET efficiency showed high values, close to 100%, for both compounds. Naked eye observation of the compounds in solution showed the typical bodipy emission (green), thereby confirming the results of this study. The emission quantum yields of this new series were not significantly affected by the presence of oxygen. For future perspective, these compounds can be applied for Organic Light Emitting Diodes (OLED's) or chemosensor devices.