Boron dipyrromethene (BODIPY) dyes bearing a pyridyl moiety have been used as metal ion sensors, pH sensors, fluorescence probes, and as sensitizers for phototherapy. A comparative study of the properties of the three structural isomers of meso-pyridyl-BODIPYs, their 2,6-dichloro derivatives, and their corresponding methylated cationic pyridinium-BODIPYs was conducted using spectroscopic and electrochemical methods, X-ray analyses, and TD-DFT calculations. Among the neutral derivatives, the 3Py and 4Py isomers showed the highest relative fluorescence quantum yields in organic solvents, which were further enhanced 2-4-fold via the introduction of two chlorines at the 2,6-positions. Among the cationic derivatives, the 2catPy showed the highest relative fluorescence quantum yield in organic solvents, which was further enhanced by the use of a bulky counter anion (PF6-). In water, the quantum yields were greatly reduced for all three isomers but were shown to be enhanced upon introduction of 2,6-dichloro groups. Our results indicate that 2,6-dichloro-meso-(2- and 3-pyridinium)-BODIPYs are the most promising for sensing applications. Furthermore, all pyridinium BODIPYs are highly water-soluble and display low cytotoxicity towards human HEp2 cells.
Boron dipyrromethene (BODIPY) dyes bearing a pyridyl moiety have been used as metal ion sensors, pH sensors, fluorescence probes, and as sensitizers for phototherapy. A comparative study of the properties of the three structural isomers of meso-pyridyl-BODIPYs, their 2,6-dichloro derivatives, and their corresponding methylated cationic pyridinium-BODIPYs was conducted using spectroscopic and electrochemical methods, X-ray analyses, and TD-DFT calculations. Among the neutral derivatives, the 3Py and 4Py isomers showed the highest relative fluorescence quantum yields in organic solvents, which were further enhanced 2-4-fold via the introduction of two chlorines at the 2,6-positions. Among the cationic derivatives, the 2catPy showed the highest relative fluorescence quantum yield in organic solvents, which was further enhanced by the use of a bulky counter anion (PF6-). In water, the quantum yields were greatly reduced for all three isomers but were shown to be enhanced upon introduction of 2,6-dichloro groups. Our results indicate that 2,6-dichloro-meso-(2- and 3-pyridinium)-BODIPYs are the most promising for sensing applications. Furthermore, all pyridinium BODIPYs are highly water-soluble and display low cytotoxicity towards human HEp2 cells.
Since the synthesis of the first boron dipyrromethene (BODIPY) in the 1960s by Treibs and Kreuzer [1], BODIPY-based dyes have attracted great attention [2,3,4,5]. This is due to their desirable properties which include large molar absorption coefficients, high photo and chemical stability [2], tunable photophysical properties [3,4], narrow spectral band width, and high fluorescence quantum yields in organic solutions [5]. BODIPYs have been developed for a wide range of applications, including photosensitizers in cancer therapy [6,7], biological labels [8,9,10,11], imaging agents [12], fluorescent switches [13,14,15], and laser dyes [16,17].While there has been a large number of BODIPY dyes designed, synthesized and characterized over the last two decades, the development and synthesis of low molecular weight and water-soluble BODIPY dyes is yet to be fully explored [18]. Additional studies in this area are needed to provide water-soluble, membrane-permeable BODIPY derivatives and widen their biological applications [19]. The water solubility of BODIPY dyes has been enhanced via installation of solubilizing groups such as phosphates [20,21], sulfonates [22,23,24,25], carboxylates [26,27,28], carbohydrates [29,30], and oligoethylene glycol chains [31]. However, these solubilizing groups increase the dye’s molecular size and tend to decrease their cell membrane permeability [32]. On the other hand, pyridinium groups are easy to install, and lead to cationic BODIPY derivatives with enhanced water-solubility, stability, and membrane permeability. In addition, meso-pyridyl-BODIPYs have been used as ligands in coordination chemistry, and as fluorescent pH sensors; therefore, this type of functionalized BODIPYs has found applications in bioimaging and biosensing. However, previous studies on a meso-(4-pyridinium)-BODIPY showed that upon excitation, the dye undergoes donor-photoinduced electron transfer (d-PeT) to a charge-transfer state that is quenched through intersystem crossing (ISC) [32]. This process occurs by the excitation (S0→S2) and subsequent non-radiative decay from (S2→S1) moving the electron from HOMO→LUMO+1→LUMO as shown in Figure 1. In this charge-transfer process, the 4-pyridinium group acts as an acceptor and the singlet-excited state of the BODIPY core acts as the donor. As a result, the fluorescence quantum yield of the dye is greatly reduced (see Figure 2a). While restoration of the meso-(4-pyridyl)-BODIPY fluorescence can be achieved by either inhibiting d-PeT or the ISC, the charge recombination emission can be enhanced to varying degrees by increasing the electrostatic charge of the ‘hole’ created by the electronic excitation and subsequent d-PeT process. We recently showed [33] that this can be accomplished through the installation of electron-withdrawing groups at the 2,6-positions. As an example, chlorination at the 2,6-positions induced an increase in the fluorescence quantum yield of a meso-4-pyridinium BODIPY from 0.019 to 0.048 in acetonitrile and from 0.004 to 0.038 in water [33]. Furthermore, Ueno and coworkers [34] reported that the installation of acetyl groups at the 2,6-positions of a meso-(nitroanisole)-BODIPY increased the fluorescence quantum yield of the dye in acetonitrile by over 20-fold (see Figure 2b).
Figure 1
Simplified Franck–Condon energy well diagram.
Figure 2
Previous work on modulating the fluorescent properties of BODIPY dyes: (a) effect of N-methylation of a meso-(4-pyridyl)-BODIPY [32], and (b) effect of introduction of acetyl groups at the 2,6-positions of a meso-aryl-BODIPY [34].
Herein we report the synthesis and investigation of meso-(2-pyridyl) and meso-(3-pyridyl)-BODIPYs, and of their 2,6-dichloro and pyridinium derivatives (see Figure 3) and compare their photophysical, electrochemical and cytotoxic properties relative to the known meso-(4-pyridyl) and meso-(4-pyridinium) derivatives and their 2,6-dichloro analogs. The moderate electron-withdrawing chlorine atoms are easily introduced at the 2,6-positions and expected to induce red-shifts in the absorption and emission wavelengths, increase reduction potentials, increase stability, and enhance the relative fluorescence quantum yields in both organic solvents and water. This type of BODIPY and halogenated derivatives could find multiple applications as optical sensors, fluorescent probes, or as sensitizers for phototherapy.
Figure 3
Structure of series of BODIPYs synthesized in this study.
2. Materials and Methods
2.1. Synthesis and Characterization
2.1.1. General
Commercially available reagents and solvents were used as received from VWR or Sigma Aldrich unless noted otherwise. All reactions were monitored by thin-layer chromatography (TLC) using 0.2 mm silica gel plates (with UV indicator, polyester backed, 60 Å, pre-coated). Liquid chromatography was performed on preparative TLC plates or via silica gel column chromatography (60 Å, 230–400 mesh). NMR spectra were measured on 400 or 500 MHz for 1H and 500 or 750 MH for 13C spectrometer. Chemical shifts (δ) are given in parts per million (ppm) In CDCl3 (7.27 ppm for 1H NMR, 77.0 ppm for 13C NMR) or (CD3)2CO (2.05 ppm for 1H NMR, 206.68 and 29.92 ppm for 13C NMR) or CD3CN (1.94(5) ppm for 1H NMR, 118.69 and 1.39 ppm for 13C NMR); coupling constants (J) are given in hertz. High-resolution mass spectra (HRMS) were obtained using an Agilent 6230-B ESI-TOF mass spectrometer.BODIPYs 2Py [35] and 3Py [36] were prepared following a procedure previously reported, by reacting 2,4-dimethylpyrrole with the corresponding pyridine 2-carbaldehyde in the presence of trifluoroacetic acid (TFA), followed by oxidation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) and complexation with BF3•(OEt)2 in triethylamine. The spectroscopic data for 2Py and 3Py are in agreement with the literature reports.
A solution of BODIPY 2Py (10.7 mg, 0.03 mmol) in dry dichloromethane (6 mL) was stirred at room temperature in an oven dried round bottom flask under N2 atmosphere. Trichloroisocyanuric acid (TCCA) (6.10 mg, 0.026 mmol) was dissolved in dichloromethane (2 mL) and added slowly to the solution. The reaction was stirred at room temperature for 2 h, until complete disappearance of the starting material. The solvent was removed under reduced pressure and the resulting residue was purified via column chromatography using 5–20% ethyl acetate in hexanes, to give 9 mg (84%) of the title compound. 1H NMR (500 MHz, CDCl3) δ 8.84 (d, J = 5.0 Hz, 1H), 7.91 (t, J = 7.7 Hz, 1H), 7.54–7.50 (m, 1H), 7.45 (d, J = 7.7 Hz, 1H), 2.61 (s, 6H), 1.32 (s, 6H). 13C NMR (126 MHz, CDCl3) δ 153.40, 153.14, 150.49, 139.18, 137.43, 137.23, 129.58, 124.39, 124.30, 29.72, 12.53, 11.36. 11B NMR (128 MHz, CDCl3) δ 0.43 (t, J = 31.7 Hz). HRMS (ESI-TOF) m/z [M+H]+ calcd for C18H16BCl2F2N3: 394.0858 found 394.0857.
This compound was prepared as described above for 3catPy, using BODIPY 3PyCl (10 mg, 0.025 mmol), anhydrous acetonitrile (10 mL), and methyl iodide (4 mL). The title compound was obtained (8.6 mg, 86% yield). 1H NMR (400 MHz, CD3CN) δ 8.94 (d, J = 6.1 Hz, 1H), 8.90 (s, 1H), 8.62 (d, J = 8.1 Hz, 1H), 8.28–8.23 (m, 1H), 4.43 (s, 3H), 2.57 (s, 6H), 1.44 (s, 6H). 13C NMR (126 MHz, CD3CN) δ 156.11, 148.72, 147.60, 145.72, 139.37, 135.25, 130.41, 50.60, 14.19. 11B NMR (128 MHz, Acetone) δ 0.55, 0.31, 0.07. HRMS (ESI-TOF) m/z [M]+ calcd for C19H19BCl2F2N3: 408.1015 found 408.1017.
2.1.8. Anion Exchange
Anion exchange was accomplished by treating the corresponding iodide cationic BODIPY (1 equivalent) in acetonitrile with excess NH4PF6 in methanol at room temperature. The mixture allowed to sit for 1 h and solvent evaporated under reduced pressure. The solid was then washed with water and the PF6 salt recovered via filtration. HRMS (ESI-TOF) m/z [M+H]+ calcd for C19H21BF2N3: 340.1794 found 340.1795 for 2catPy. HRMS (ESI-TOF) m/z [M+H]+ calcd for C19H21BF2N3: 340.1794 found 340.1795 for 3catPy. HRMS (ESI-TOF) m/z [M]+ calcd for C19H21BF2N3: 339.1827 found 339.1827 for 4catPy. HRMS (ESI-TOF) m/z [M]+ calcd for C19H19BCl2F2N3: 408.1015 found 408.1017 for 2catPyCl. HRMS (ESI-TOF) m/z [M]+ calcd for C19H19BCl2F2N3: 408.1015 found 408.1015 for 3catPyCl. HRMS (ESI-TOF) m/z [M]+ calcd for C19H19BCl2F2N3: 408.1015 found 408.1015 for 4catPyCl.
2.2. Spectroscopy Methods
UV−vis absorption spectra were collected on a Varian Cary 50 Bio spectrophotometer. Emission spectra were obtained on a PerkinElmer LS55 spectrophotometer, at room temperature. Spectrophotometric grade solvents and quartz cuvettes (1 cm path length) were used. Relative fluorescence quantum yields (Φf) were calculated using rhodamine 6G (Φf = 0.86 in methanol) as reference for the neutral BODIPYs and Ru(bpy)Cl2.6H2O (Φf = 0.028 in water) as the standard for the cationic BODIPYs using the following equation: Φx = Φst × Gradx/Gradst × (ηx/ηst)2, where the ΦX and ΦST are the quantum yields of the sample and standard, GradX and GradST are the gradients from the plot of integrated fluorescence intensity vs. absorbance, and η represents the refractive index of the solvent (x is for the sample and st for the standard).
2.3. Electrochemistry
Cyclic voltammograms were collected on a Biologic SP-300 potentiostat using a three-electrode setup with a platinum working electrode (2 mm diameter, CH Instruments), platinum counter electrode (2 mm diameter, CH Instruments), and a silver wire pseudo reference-electrode stored in 0.25 M NBu4PF6 in anhydrous acetonitrile and separated from the electrochemical cell by a glass frit. The electrochemical cell was composed of a borosilicate glass scintillation vial with a custom-machined cap. Working electrodes were polished and then pre-treated by cycling between upper and lower boundaries of the electrochemical window for the experiment in a solution containing only the electrolyte. Measurements were taken in a solution of 0.25 M recrystallized NBu4PF6 in dried and degassed anhydrous acetonitrile. BODIPYs were added to 5 mL of electrolyte solution to give a concentration of 1 mM. Each solution also contained 1 mM ferrocene as an internal reference. Solutions were sparged with N2 before each measurement.
2.4. X-ray Crystallography
The crystal structures of 2Py, 2PyCl and 3Py were determined at T = 90 K from data collected on a Bruker Kappa Apex-II diffractometer equipped with Mo Kα (for 2Py and 2PyCl) or CuKα (for 3Py) source. Absorption corrections were by the multi-scan method. Non-hydrogen atoms were treated with anisotropic displacement parameters. The H atoms were visible in difference maps and were placed in idealized positions in the refinements, with torsional parameters refined for methyl groups. For 2Py and 2PyCl, disorder exists in which the pyridyl group is rotated 180° about the bond joining it to the BODIPY core. Crystal Data: 2Py, C18H18BF2N3, FW = 325.16, orthorhombic space group Pbca, a = 12.7201(8), b = 12.2790(8), c = 20.1538(13) Å, V = 3147.8(3) Å3, Z = 8, θmax = 38.6°, R = 0.054 for the 5941 I > 2σ(I) data, wR(F2) = 0.146 for all 8744 data and 222 parameters. The CIF has been deposited at the Cambridge Crystallographic Data Centre, CCDC 2163983; 2PyCl2, C18H16BCl2F2N3, FW = 394.05, monoclinic space group P21/c, a = 6.4405(8), b = 15.6937(19), c = 17.205(2) Å, β = 93.150(4)°, V = 1736.4(4)Å3, Z = 4, θmax = 29.2°, R = 0.067 for the 3122 I > 2σ(I) data, wR(F2) = 0.126 for all 4694 data and 240 parameters, CCDC 2163984; 3Py, C18H18BF2N3, FW = 325.16, monoclinic space group P21, a = 7.0759(2), b = 12.0387(3), c = 19.0447(5) Å, β = 96.182(2)°, V = 1612.88(7)Å3, Z = 4, θmax = 69.5°, R = 0.030 for the 5618 I > 2σ(I) data, wR(F2) = 0.073 for all 6017 data and 441 parameters, Flack x = −0.03(5), CCDC 2163985.
2.5. Computational Methods
The geometries of the ground and excited states of all compounds were optimized without symmetry constraints at the cam-b3lyp/6-31+G(d,p) level in vacuum. This method has been shown to correctly reproduce the experimental trends [18]. The UV-vis absorption data were calculated using the TD-DFT method at the same level. The rotational barriers were calculated in CH3CN taking the solvent effects into account using the Polarized Continuum Model (PCM). All calculations were performed using the Gaussian 09 program package [37].
2.6. Cell Toxicity
Human carcinoma HEp2 cells were obtained from ATCC and maintained in a 75 cm2 flask (Chemglass) with 1× MEM (Eagle’s minimal essential medium) containing 10% FBS (fetal bovine serum) and 1% antibiotic (penicillin–streptomycin) in a humidified, 5% CO2 incubator at 37 °C. Stock solutions with a concentration of 32 mM in DMSO were prepared for each BODIPY. The stock solution was diluted to the final working concentrations with culture medium. The HEp2 cells were plated at 7500–10,000 cells per well in a Costar 96-well plate and allowed to grow for 24 h. The BODIPY stock solution was diluted to a 200 μM working solution and two-fold serial dilutions were prepared with final concentrations of 200, 100, 50, 25, 12.5, 6.25, and 0 μM. Each BODIPY was incubated for 24 h at 37 °C under 5% CO2. For the dark cytotoxicity experiments, after incubation, excess BODIPY was removed by washing the cells three times with 1× PBS (phosphate-buffered saline) and replacing with media containing 20% Cell Titer Blue (Promega, Madison, WI, USA). The cells were then incubated for additional 24 h at 37 °C under 5% CO2 and the cell toxicity was determined by fluorescence intensity at ex 570/em 615 nm using a FluoStar Optima microplate reader (BMG Labtech, Cary, NC, USA).For the phototoxicity experiments, after incubation the cells were exposed to a light dose of ~1.5 J/cm2 generated using a 600 W Quartz Tungsten Halogen lamp (Newport Corporation, Irvine, CA, USA). The cells were exposed for 20 min while the 96-well plate rested on an EchoTherm chilling plate (Torrey Pines Scientific, Carlsbad, CA, USA) set to 5 °C. Following light exposure, the loading media was removed and media containing 20% Cell Titer Blue was added. After incubation for 24 h at 37 °C under 5% CO2, the cell viability was determined as described above.
3. Results and Discussion
3.1. Synthesis
The 2Py [35] and 3Py [36] BODIPYs were prepared in 48% and 55% yields, respectively, by condensation of 2,4-dimethylpyrrole with the corresponding pyridine carboxaldehyde in presence of TFA, followed by oxidation with DDQ and boron complexation using boron trifluoride etherate, as previously reported. Treating BODIPYs 2Py and 3Py with excess of methyl iodide in acetonitrile gave the corresponding pyridinium BODIPYs, 2catPy and 3catPy, in nearly quantitative yields. Anion exchange was performed on these BODIPYs using ammonium hexafluorophosphate (NH4PF6). The resulting salts were purified by washing with organic solvents and characterized by mass spectrometry.Chlorination of BODIPYs 2Py and 3Py using TCCA in dichloromethane [33] produced the corresponding 2,6-dichloro derivatives 2PyCl and 3PyCl in 84% and 89% yields, respectively. Methylation of these 2,6-dichloro-BODIPYs using methyl iodide afforded their pyridinium derivatives 2catPyCl and 3catPyCl. Anion exchange was performed on these BODIPYs using ammonium hexafluorophosphate. The structures of all the BODIPYs were confirmed by 1H, 13C and 11B NMR (see Supplementary Materials, Figures S3–S26) and by high-resolution mass spectrometry (ESI-TOF).
3.2. X-ray Analysis
Crystals of 2Py, 3Py and 2PyCl were obtained by slow evaporation of dichloromethane and pentane, and the results are shown in Figure 4. BODIPYs 2Py, 3Py, and 2PyCl all have their 12-atom BODIPY cores nearly planar, with mean deviations from coplanarity of 0.029, 0.023 and 0.038 Å, respectively. The dihedral angles between the BODIPY core planes and the pyridyl planes are also similar, 79.3° for 2Py, 82.3° for 3Py (average of two independent molecules), and 85.9° for 2PyCl. These values agree well with those for the published structure of 4Py [, for which the mean deviation from the BODIPY core is 0.018 Å and the BODIPY/Py dihedral angle is 83.9° (average of two). Bond distances and angles also show excellent agreement across the three structures and in agreement with the published structure of 3Py [36]. The relatively lower dihedral angle observed for 2Py relative to the 3Py and 4Py suggests a slight increased flexibility for 2Py, although the difference is very small. Therefore, for all three isomers the pyridine is interlocked between the 1,7-methyl substituents and does not affect the molecular structure of the BODIPY core.
Figure 4
Crystal structures of 2Py, 2PyCl and 3Py BODIPYs with 50% ellipsoids.
3.3. Spectroscopic Properties
The absorption and emission spectra of BODIPYs 2Py, 3Py, 2PyCl and their cationic derivatives 2catPy, 3catPy, 2catPyCl were obtained at room temperature in acetonitrile, methanol, and water, and the results are as summarized in Table 1, and in Figure S1 and Table S1 of the Supplementary Materials. As expected, the absorption spectra for 2Py, 3Py and 4Py featured a sharp peak at ca. 501 nm which is attributed to the BODIPYs strong S0-S1 (π-π*) transition, as confirmed by molecular modeling. The broad high energy shoulder bands are attributed to the BODIPYs vibrational transitions. The emission peak for these BODIPYs is seen at ca. 514 nm, and the relatively small Stokes shift indicates that the molecular structure of the excited state is not very different than that of the ground state. The maximum absorption and emission wavelengths for the neutral 2Py, 3Py and 4Py are almost identical in both acetonitrile and methanol (see Table 1 and Table S1). In agreement with this observation, computational modeling of the absorption spectra in the gas phase confirms the almost identical maximum absorption wavelengths, which correspond well to the almost identical HOMO-LUMO gaps, as can be seen in Table 2. For the entire series of 2Py, 3Py and 4Py, the HOMO is almost entirely localized on the BODIPY core, whereas the LUMO partially involves the pyridyl group (see Figure 5a, and Figure A1 and Figure A2 of Appendix A). LUMO+1 is predominantly on the pyridyl group.
Table 1
Spectroscopic properties of BODIPYs in CH3CN and H2O and relative fluorescence quantum yields.
Solvent
BODIPY
λabs (nm)
λem (nm)
Stokes Shift (nm)
Φf
ᵋ (M−1 cm −1)
CH3CN
2Py
502
514
12
0.04 a
87,500
3Py
502
514
12
0.43 a
92,900
4Py [18]
501
515
14
0.31
72,100
2PyCl2
530
545
15
0.17 a
64,280
3PyCl2
527
542
15
0.58 a
57,540
4PyCl2 [18]
528
546
18
0.58
50,800
2catPy (I−)
520
544
24
0.13 b
65,800
2catPy (PF6−)
520
546
26
0.23 b
36,700
3catPy (I−)
510
532
22
0.006 b
65,460
3catPy (PF6−)
510
529
19
0.006 b
88,580
4catPy (I−) [18]
509
596
87
0.019
26,000
4catPy (PF6−)
509
603
94
0.010 b
71,520
2catPyCl2 (I−)
530
547
17
0.17 b
19,420
3catPyCl2 (I−)
540
560
20
0.23 b
43,160
4catPyCl2 (I−) [18]
538
600
62
0.048
23,400
H2O
2catPy (I−)
520
542
22
0.001 b
52,380
3catPy (I−)
510
530
20
0.005 b
37,320
4catPy (I−) [18]
509
600
91
0.004
31,600
2catPyCl2 (I−)
555
577
22
0.24 b
21,000
3catPyCl2 (I−)
540
564
24
0.47 b
35,180
4catPyCl2 (I−)
540
605
65
0.038
11,640
a Calculated using rhodamine 6G (Φf = 0.86) in methanol at λexc = 473 nm as the standard. b Calculated using Ru(bpy)Cl2.6H2O (Φf = 0.028) in H2O at λexc = 436 nm as the standard.
Table 2
Calculated spectroscopic properties, HOMO-LUMOBODIPY gap, rotational barriers of the pyridyl or pyridinium groups with respect to the BODIPY core, and S1→T energy difference (cam-b3lyp/6-31+G(d,p) in gas phase). The LUMOBODIPY is LUMO for the neutral species but LUMO+1 for the cationic species.
BODIPY
λabs (nm)
Oscillator Strength
HOMO-LUMOBODIPY Gap (eV)
RotationBarrier (kcal/mol)
S1-T Energy Difference (eV)
2Py
416
0.542
5.14
17.8
3Py
416
0.542
5.14
20.7
4Py
415
0.544
5.15
19.7
2catPy
435
0.537
4.88
>35
0.057
3catPy
429
0.490
4.95
25.3
0.025
4catPy
427
0.550
4.98
25.1
0.012
2PyCl2
432
0.565
5.05
16.6
3PyCl2
433
0.562
5.03
18.6
4PyCl2
432
0.561
5.04
19.2
2catPyCl2
460
0.558
4.72
-
3catPyCl2
455
0.533
4.78
-
4catPyCl2
451
0.567
4.82
-
Figure 5
Frontier orbitals of (a) 3Py, (b) 3catPy, (c) 3PyCl and (d) 3catPyCl BODIPYs. Orbital energies in eV. The frontier orbitals for the entire series are given in Appendix A.
Figure A1
Frontier orbitals of (a) 2Py, (b) 2catPy, (c) 2PyCl and (d) 2catPyCl BODIPYs. Orbital energies in eV.
Figure A2
Frontier orbitals of (a) 4Py, (b) 4catPy, (c) 4PyCl and (d) 4catPyCl BODIPYs. Orbital energies in eV.
The presence of a 2-, 3- or 4-pyridyl group at the meso-position has a strong effect on the relative fluorescence quantum yields using rhodamine 6G as the reference (Φf = 0.86 in methanol) [40]. BODIPY 3Py was found to be the most fluorescent (Φf = 0.43) and the 2Py is the least (Φf = 0.04). The high fluorescence of 3Py and the 4Py in organic solvents has previously been observed [36,41,42]. On the other hand, the 2Py displays much lower fluorescence compared with its regioisomers, as previously reported [35,43]. This is due to the closer proximity of the nitrogen atom to the BODIPY core in 2Py relative to the 3Py and 4Py compounds. The 2-pyridyl group has a lower rotational barrier compared with the 3- and 4-pyridyl groups (see Table 2), suggesting that in the case of 2Py more energy is released via non-radiative decay pathways. The meso-pyridyl rotation causes distortion of the BODIPY core that disrupts electron conjugation and could be the reason for the observed lower quantum yields for 2Py. Another possible explanation is the difference in the S1 LUMO. In the case of 3Py and the 4Py, the S1 LUMO is almost entirely localized on BODIPY, whereas for 2Py, the orbital is delocalized over BODIPY and pyridyl.Methylation of the pyridyl group leads to a decrease in the molar extinction coefficients, as well as bathochromic shifts in the maximum absorption wavelengths of the cationic derivatives in acetonitrile, that was more pronounced for the 2catPy (18 nm) relative to the 3catPy (8 nm) and 4catPy (8 nm). This tendency is also seen in the calculated maximum absorption wavelengths; all cationic BODIPYs show bathochromic shifts with that of 2catPy (19 nm) being more pronounced than for 3catPy (13 nm) and 4catPy (12 nm), demonstrating similar shifts. For this series of methylated compounds, the HOMO is again almost entirely located on the BODIPY core but the LUMO and LUMO+1 switch compared with the non-methylated analogs. In this case, LUMO is mostly on the pyridinium group, whereas LUMO+1 is mostly on BODIPY core (see Figure 5 and Figure A1 and Figure A2 of Appendix A). This suggests that PeT occurs from the BODIPY core to the pyridinium group upon excitation, which quenches a high percentage of the fluorescence. The gaps between HOMO and LUMO+1 (LUMOBODIPY) are given in Table 2, as this is the transition corresponding to the fluorescence activity. The electron-withdrawing effect lowers both HOMO and LUMOBODIPY with greater effect on LUMOBODIPY, thus decreasing the gap between these two orbitals, which is in agreement with the experimentally observed bathochromic shift.The observed bathochromic shifts are similar in the case of 3catPy and 4catPy but different in the case of 2catPy. This is likely due to the stronger electron-withdrawing effect of the 2-pyridinium group due to the proximity of the nitrogen atom to the BODIPY core. While in the case of the 3catPy and 4catPy the pyridinium nitrogen pulls electron density nearly equally from both sides of the BODIPY core, in the case of 2catPy there is a direct pull of electron density from the nearby carbon-2 of the pyridyl group, which induces the observed enhanced bathochromic shift. This is in agreement with the observed similar orbital energies of HOMO and LUMOBODIPY in the case of 3catPy and 4catPy, and lower orbital energies in the case 2catPy (see Figure 5 and Figure A1 and Figure A2 of Appendix A). Furthermore, moderate redshifted emissions were observed for 2catPy and 3catPy, centered at 544 and 532 nm, respectively. In the case of 4catPy a more pronounced redshift to 596 nm was observed, as we previously reported [33], likely due to the highly dipolar species formed upon excitation, with a pronounced charge-transfer character [44].The fluorescence in acetonitrile of 3catPy was drastically reduced to 0.006, as previously observed for 4catPy [33], due to intramolecular charge transfer, since pyridinium is more easily reduced than pyridine. This has led to the exploration of photo- or chemical induced dealkylation of a 4-pyridinium quencher as a strategy to restore the fluorescence of the BODIPY chromophore [45,46,47]. On the other hand, the fluorescence of 2catPy actually increased to 0.04 to 0.13 or 0.23 with I− or PF6− as the counter ion, respectively. The observed lower quantum yields for 3catPy and 4catPy compared with 3Py and 4Py are likely due to intramolecular charge transfer, since pyridinium is more easily reduced than pyridine. The energy difference between LUMO and LUMO+1 is only 0.30 eV in the case of 2catPy and 0.79/0.89 eV in 3catPy/4catPy, respectively (see Figure 5a, and Figure A1 and Figure A2 of Appendix A), suggesting easier charge-recombination and higher quantum yield in 2catPy. Furthermore, 3catPy and 4catPy show higher probability for intersystem crossing compared with 2catPy, as suggested by the lower S1→T energy differences (Table 2). The increase in fluorescence upon alkylation of 2Py has been previously observed [35], and is in part due to the bulkiness of the alkyl group on the pyridinium nitrogen atom in close proximity to the BODIPY core. The larger counter ion (PF6−) induces the highest relative fluorescence in the 2catPy, but this effect is not observed for the 3catPy nor the 4catPy. We hypothesize that in the case of the cationic 3- and 4-pyridinium-BODIPYs with iodide as counter ion, there is likely a close interaction of the BODIPYs with the iodide anion, since the pyridinium nitrogen in 3catPy and 4catPy is easily accessible. Therefore, the iodide anion in these cases is able to exert a heavy-atom effect that leads to ISC from the lowest singlet excited state (S1) to the triplet state of the cationic system [48]. On the other hand, in BODIPY 2catPy the sterically hindered cationic nitrogen center prevents close interaction with the iodide ion.In water, the fluorescence quantum yields decreased further due to the increase in solvent polarity which stabilizes the charge-transfer state leading to deactivation through ISC, as previously observed for 2catPy [35] and 4catPy [33]. However, the maximum absorption and emission observed for the 2catPy and 3catPy remained unchanged in acetonitrile, methanol, and water (see Table 1 and Supplementary Table S1 in Supplementary Materials).The installation of the 2,6-dichloro substituents led to significant redshifted absorptions (25–27 nm), decreased molar absorptivities, red-shifted emissions (28–31 nm), and slight increased Stokes shifts of the neutral BODIPYs, as previously observed [33,49], due to the electron-withdrawing effect of the chlorine atoms. The chlorines at the 2,6-positions stabilize both the HOMO and LUMO, and this effect is more pronounced on the LUMO (see Figure 5 and Figure A1 and Figure A2 of Appendix A). As a result, the HOMO-LUMO gaps are smaller for the chlorinated derivatives, causing the observed redshifts of the absorption wavelengths. The performed calculations show an increase in the oscillator strengths upon introduction of the chlorine atoms. In addition, the experimental relative fluorescence quantum yields increased upon 2,6-dichlorination, likely due to reduction in nonradiative deactivation, as it was previously shown that the nonradiative rate constant significantly decreases with the introduction of chlorine groups [49]. Chlorination at the 2,6-positions also increased the fluorescence quantum yields of the BODIPYs due to the decrease in electronic charge density on the BODIPYs. Compound 2PyCl again showed relatively reduced fluorescence (Φf = 0.17) in comparison with 3PyCl and 4PyCl (both Φf = 0.58) due to its closer proximity of the nitrogen atom to the BODIPY core.The corresponding methylated 2,6-dichlorinated analogues showed increase in fluorescence quantum yields in acetonitrile, particularly in the case of 3catPyCl which showed a 38-fold enhancement in fluorescence relative to 3catPy In water, the absorption and emission wavelengths of the cationic derivatives did not change significantly, except in the case of 2catPyCl. Furthermore, the fluorescence quantum yields were partially enhanced upon introduction of the 2,6-chlorines, as we previously reported in the case of 4catPyCl [33]. A drastic increase was observed in the case of 2catPyCl (Φf = 0.24) which displayed over 200-fold increase in fluorescence relative to 2catPy, whereas 100- and 10-fold enhancements were observed for 3catPyCl and 4catPyCl, respectively. The observed fluorescence enhancements are likely due to an alteration of the excited state lifetime resulting from the increase in the BODIPY oxidation potential of the chlorinated BODIPY derivatives in addition to the naturally higher oxidation potential of the 2-pyridinium BODIPY (see below), which increases the rate of charge recombination by magnifying the electron-hole electrostatic interaction. In agreement with these findings, up to 100-fold fluorescence enhancement was reported for a 2catPy derivative bearing methylmercaptophenoxy groups in place of the fluorines on the boron atom, upon oxidation to the corresponding sulfoxides [50]. Furthermore, our results show that the fluorescence of 3catPyCl in water (Φf = 0.47) is the highest among the series of pyridinium-BODIPYs.
3.4. Electrochemical Properties
The electrochemical data for the pyridinium-BODIPYs 2catPy, 3catPy and 4catPy with PF6− as counterion are summarized in Table 3 and shown in Figure 6. Each pyridinium-BODIPY shows one quasi-reversible oxidation corresponding to the formation of the p-radical cation [51]. Due to the stronger electron-withdrawing effect of the 2-pyridinium group in 2catPy, the oxidation potential is shifted positive by 100 mV compared with 4catPy and 3catPy. Each pyridinium-BODIPY shows reversible reductions that occur either through an apparent two-electron reduction or two separate single-electron reductions. 4catPy undergoes a single two-electron reduction which corresponds to the reductions localized on the BODIPY core and the pyridinium group. It is likely that the initial reduction forms a radical which is more easily reduced than the initial BODIPY compound, resulting in the second electron transfer occurring almost immediately upon the formation of the neutral radical. These results are in agreement with previous published data [32]. Meanwhile, 2catPy and 3catPy show two sequential reversible one electron reductions, which suggest an increased stability of the radical formed upon the initial reduction (Figure 6a). Overall, 2catPy and 3catPy are more resistant to reduction to either the singly or doubly reduced state compared with 4catPy. BODIPY 2catPy undergoes its first reduction 30 mV more negative and its second reduction 230 mV more negative than the two-electron reduction seen in 4catPy. BODIPY 3catPy is more significantly affected as its first reduction appears 200 mV more negative and its second reduction appears 480 mV more negative than the two-electron reduction in 4catPy. The first reduction for both 2catPy and 3catPy lies in the potential range usually seen for the reduction in the BODIPY core to its singly reduced state, whereas the second reduction may correspond to the reduction in the pyridinium group [51]. Thus, the influence of meso-pyridinium substituents on the BODIPY redox potentials are moderate (~170 mV) and consistent with the electron-withdrawing effects of the pyridinium groups. On the other hand, the introduction of the 2,6-dichloro groups induces larger effects, as discussed below.
Table 3
Oxidation and reduction potentials for BODIPYs determined by cyclic voltammetry (in V vs. Fc+/Fc) at 100 mV/s in degassed and anhydrous acetonitrile with 0.25 M NBu4PF6 at analyte concentrations of 0.001 M.
BODIPY
E1/2ox(V)
E1/2red(V)
2catPy
+0.99 a
−1.09; −1.29
3catPy
+0.89
−1.26; −1.54
4catPy
+0.89
−1.06 e
2catPyCl2
+1.10 b
−0.87; −1.16
3catPyCl2
+1.10 c
−1.02; −1.42
4catPyCl2
+1.03 d
−0.89 e
a Value estimated: this wave is more irreversible than the others. b Value at 10 V/s. c Value for oxidation peak potential only: this wave is irreversible. d Value at 1 V/s. e Two-electron reduction to doubly reduced state.
Figure 6
Cyclic voltammograms at 100 mV/s on platinum electrodes of 1 mM solutions of (a) 2catPy (blue), 3catPy (red), and 4catPy (black) and (b) 2catPyCl (blue), 3catPyCl (red), and 4catPyCl (black), in degassed, anhydrous acetonitrile with 0.25 M NBu4PF6 supporting electrolyte. Arrows indicate the direction of the potential scan. The wave marked with an asterisk (*) at 0 V vs. Fc+/Fc corresponds to the internal Fc standard.
Similar trends were seen with chlorinated versions 2catPyCl, 3catPyCl, and 4catPyCl (Figure 6b and Table 3). The 2,6-dichlorinated BODIPYs have the same number of redox events as their unchlorinated counterparts but the reduction and oxidation waves are shifted positive. The oxidation wave is irreversible for each at 100 mV/s but becomes more reversible at 1 V/s for 4catPyCl, and at 10 V/s for 2catPyCl. Compared with the corresponding unchlorinated BODIPYs at 100 mV/s, the anodic peak for each was shifted positive by 70 mV, 160 mV, and 180 mV for 2catPyCl, 3catPyCl, and 4catPyCl, respectively. As in 4catPy, 4catPyCl undergoes a reversible two-electron reduction, which is shifted 180 mV positive compared with 4catPy. Meanwhile 2catPyCl and 3catPyCl undergo two sequential one-electron reductions. The first reduction wave is reversible for both 2catPyCl and 3catPyCl and is shifted positive by 220 mV and 240 mV, respectively, when compared with their unchlorinated BODIPYs. The second, more negative reduction wave, is reversible in 2catPyCl but quasi-reversible in 3catPyCl and is shifted positive by 130 mV and 120 mV when compared with 2catPy and 3catPy, respectively. As expected, the introduction of the 2,6-dichloro groups has a larger influence on the electrochemical properties of BODIPYs than the meso-pyridinium substituents.
3.5. Cytotoxicity Properties
The dark and photocytoxicity of this series of BODIPYs was investigated in human carcinoma HEp2 cells and the results are summarized in Table 4 and shown in Figure S2 of the Supplementary Materials. Very low cytotoxicity was observed for all the BODIPYs without 2,6-chlorine substitution (IC50 > 200 mM in the dark and >100 mM at 1.5 J/cm2). The neutral 2,6-dichloro derivatives showed slightly enhanced cytotoxicity, particularly the 3PyCl due to the introduction of the chlorine atoms. The most cytotoxic were found to be the cationic 2,6-dichloro derivatives 2catPyCl and 3catPyCl. These results are in agreement with previous observations showing that the introduction of heavier halogens at the 2,6-positions, such as iodides, drastically reduce the fluorescence and increase the phototoxicity of meso-(4-pyridinium)-BODIPYs toward human cancer cells [38] and pathogenic microorganisms [52,53,54].
Table 4
Dark and photo (1.5 J/cm2) cytotoxicity of BODIPYs toward HEp2 cells.
BODIPY
Dark Toxicity(IC50, mM)
Phototoxicity(IC50, mM)
2Py
>200
>100
3Py
>200
>100
4Py
>200
>100
2PyCl2
>200
>100
3PyCl2
200
>100
2catPy
>200
>100
3catPy
>200
>100
4catPy
>200
>100
2catPyCl2
80
80
3catPyCl2
120
>100
4. Conclusions
The photophysical, electrochemical, and cytotoxic properties of meso-(2-pyridyl) and meso-(3-pyridyl)-BODIPYs, and their 2,6-dichloro and pyridinium derivatives were compared with those of their corresponding meso-(4-pyridyl) and meso-(4-pyridinium) analogs. Due to the stronger electron-withdrawing effect of the 2-pyridinium group in 2catPy, its oxidation potential was shifted positive by 100 mV compared with 4catPy and 3catPy. BODIPYs 2catPy and 3catPy were found to be more resistant to reduction compared with 4catPy. Unlike the presence of heavy halogens (Br, I) at the BODIPY 2,6-positions, which is known to enhance ISC, the presence of 2,6-chlorines was shown to enhance the relative fluorescence quantum yields of the BODIPYs in both organic solvents and water. Furthermore, the 2catPy isomer showed the highest relative fluorescence quantum yield in organic solvents, which was further enhanced by the use of a bulky counter anion (PF6−). In water, over a 200-fold increase in fluorescence was observed for 2catPyCl, 100-fold increase for 3catPyCl and 10-fold increase for 4catPyCl. The fluorescence of 3catPyCl in water (Ff = 0.47) was found to be the highest among the series of BODIPYs. Our results show that the introduction of 2,6-chlorines in BODIPYs has a larger influence on their electrochemical properties than the meso-pyridyl substituents.
Authors: M J Ortiz; I Garcia-Moreno; A R Agarrabeitia; G Duran-Sampedro; A Costela; R Sastre; F López Arbeloa; J Bañuelos Prieto; I López Arbeloa Journal: Phys Chem Chem Phys Date: 2010-05-25 Impact factor: 3.676
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Figure A1
Figure A2
Figure 6