Accessing near-infrared-absorbing BF2-azadipyrromethenes via a push-pull effect

Article abstract of DOI:10.1021/jo402160b

Novel aza-BODIPYs with significant bathochromic shifts were designed and synthesized by installation of strong electron-withdrawing groups on the para-positions of 1,7-phenyls and electron-donating groups on the para-positions of 3,4-phenyls. These dyes s

Full text of DOI:10.1021/jo402160b

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Note
Accessing Near-Infrared Absorbing BF2-
Azadipyrromethenes via a Push-Pull Effect
Lijuan Jiao, Yayang Wu, Sufan Wang, Xiaoke Hu, Ping Zhang, Changjiang
Yu, Kebing Cong, Qianli Meng, Erhong Hao, and M. Graca Henriques Vicente
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/jo402160b • Publication Date (Web): 29 Jan 2014
Downloaded from http://pubs.acs.org on February 5, 2014
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Accessing NearꢀInfrared Absorbing BF2ꢀ
Azadipyrromethenes via a PushꢀPull Effect
Lijuan Jiao,a* Yayang Wu,a Sufan Wang,a* Xiaoke Hu,b Ping Zhang,a Changjiang Yu,a Kebing Cong,a
Qianli Meng,b Erhong Haoa and M. Graça H.Vicenteb*
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aLaboratory of Functional Molecular Solids, Ministry of Education; Anhui Laboratory of Moleculeꢀ
Based Materials; School of Chemistry and Materials Science, Anhui Normal University, Wuhu, Anhui,
China 241000.
bDepartment of Chemistry, Louisiana State University, Baton Rouge, LA 70803. Fax: (+) 225-578-7405
*To whom correspondence should be addressed. Eꢀmail: jiao421@mail.ahnu.edu.cn,
sfwang@mail.ahnu.edu.cn, vicente@lsu.edu
Red shift
NC
CN
N
B
N
B
N
N
N
N
F
F
F
F
MeO
OMe
MeO
OMe
max (CHCl3) = 716 nm
em (CHCl3) = 755 nm
max (CHCl3) = 688 nm
em (CHCl3) = 723 nm
Abstract: Novel azaꢀBODIPYs with significant bathochromic shifts were designed and synthesized by
installation of strong electronꢀwithdrawing groups on the para-positions of 1,7ꢀphenyls. These dyes
show strong NIR fluorescence emissions up to 756 nm, with absorptions up to 720 nm.
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Fluorescent dyes with absorptions and emissions in the nearꢀinfrared (NIR) region have found important
applications in biology and medicine sciences1, such as sensing and imaging since NIR light penetrates
deeper into most biological tissues than visible light.2 Azadipyrromethenes boron difluoride (known as
azaꢀBODIPYs), which typically show an around 90 nm redꢀshift of the main absorption band with
respect to that of their BODIPY analogues,3,4 have recently received much attention due to their
remarkable photochemical properties,5 and have found various applications in photovoltaics,
optoelectronics, bioimaging, sensing, and photodynamic therapy.5ꢀ7
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N
B
N
B
1
3
N
B
7
N
N
N
N
N
N
F
F
5
O O
F
F
MeO
OMe
MeO
OMe
A1
B
C
max (CHCl3) = 650 nm
em (CHCl3) = 682 nm
max (CHCl3) = 740 nm
em (CHCl3) = 751 nm
max (CHCl3) = 765 nm
em (CHCl3) = 782 nm
N
N
N
N
N
N
B
B
F
F
F
F
N
N
B
BDP1
F
F
max (CH3OH) = 528 nm
em (CH3OH) = 552 nm
D
MeO
OMe
max (CH2Cl2) = 729 nm
em (CH2Cl2) = 769 nm
F
CN
N
Hex
Hex
Hex
Hex
N
N
N
N
B
B
F
F
F
F
S
S
BDP2
E
NO2
O2N
max (CH3OH) = 588 nm
em (CH3OH) = 612 nm
max (CH2Cl2) = 717 nm
em (CH2Cl2) = 739 nm
max (CH2Cl2) = 694 nm
em (CH2Cl2) = 741 nm
Figure 1. Reported strategies aimed at extending the absorption and emissiom of azaꢀBODIPYs to the
NIR spectral range.
The parent 1,3,5,7ꢀtetraphenylꢀazaꢀBODIPY A1 (Fig. 1) absorbs at 650 nm and emits at 682 nm in
CHCl3. Several studies have focused on the development of new azaꢀBODIPY dyes with redꢀshifted
absorptions and emissions further to the NIR range. Various elegant approaches have been reported,
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include: (i) rigidification of rotatable moieties, as demonstrated by azaꢀBODIPYs B8 and C,9 (ii)
planarization of the πꢀsystem through reducing the torsion angles, as demonstrated by replacing 3,5 ꢀ
1
2
3
4
phenyls with thiophenes in azaꢀBODIPY E,10 (iii) extension of the πꢀconjugation, as demonstrated by
the benzeneꢀfused azaꢀBODIPY D,11 and by azaꢀBODIPY F.12 However, the syntheses of these
compounds generally require multiple steps with lower yields in comparison to that of the parent
compound A1, which limit their practical applications. On the other hand, the simple introduction of
electronꢀdonating groups on the paraꢀpositions of 3,5ꢀphenyls results in significant redꢀshifts of both
absorption and emission bands. For example, azaꢀBODIPY A2 (Scheme 1) bearing electronꢀdonating
methoxy groups (Hammett parameter σp = ꢀ0.27) absorbs at 680 nm and emits at 723 nm in CHCl3,
giving a 38 and 41 nm redꢀshifts in absorption and emission, respectively, in comparison to azaꢀ
BODIPY A1. The reducing of the band gap by increasing the HOMO and/or decreasing the LUMO
energy is crucial for the development of longer wavelength azaꢀBODIPYs.11 The attachment of an
electronꢀwithdrawing group may reduce the LUMO energy. For example, BDP2 bearing a strong
electronꢀwithdrawing cyano group at the mesoꢀposition, shows a 60 nm redꢀshift of absorption with
respect to BDP1 (Fig. 1).3a,13 By contrast, BODIPYs with electronꢀdonating methylamino and methoxy
groups on the mesoꢀposition, exhibit about 51 and 85 nm blue shifts of the absorption bands due to the
increase of the LUMO energy.14 Based on these considerations, azaꢀBODIPYs A3, A4 and A6 bearing
strong electronꢀwithdrawing groups at the paraꢀpositions of 1,7ꢀphenyls were designed, synthesized,
characterized and compared with their reference compounds: azaꢀBODIPYs A1 and A2.
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Table 1. DFT Calculation Results
f b
λabs a (nm)
598
dyes excited state
orbital
H→L
HOMO/LUMO (eV)
ꢀ5.665/ꢀ3.465
S1
S3
S1
S3
0.8490
0.3882
0.8575
0.6610
A1
482
Hꢀ2→L
H→L
647
ꢀ5.380/ꢀ3.337
A2
485
Hꢀ1→L
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S1
S3
687
495
ꢀ5.574/ꢀ3.645
0.7946
0.4533
A4
H→L
1
2
3
Hꢀ1→L
a Stands for calculated absorption peaks. b Oscillator strength.
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O
O
KOH
MeO
+
R1
CHO
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MeO
1a R = CF3
1b R = CN
1c R = N(CH3)2
R
MeO
R1
1) NH4OAc
EtOH
CH3NO2
A3-A5
O
NHEt2
MeOH
2) BF3.Et2O
NEt3
NO2
2a R = CF3
2b
R = CN
2c R = N(CH3)2
NC
CN
F3C
CF3
N
N
N
B
N
N
N
N
N
N
B
B
F
F
F
F
F
F
MeO
OMe
MeO
OMe
OMe
MeO
N
A2
A3
A4
N
N
N
2 I
N
B
CH3I
N
N
N
N
N
B
F
F
F
F
A5
A6
MeO
OMe
MeO
OMe
Scheme 1. Syntheses of azaꢀBODIPYs A1-A7.
Initially, DFT calculations on azaꢀBODIPYs A1, A2 and A4 were carried out for the evaluation of our
hypothesis and the results are summarized in Table 1 and in Figs. S1 and S2 in the supporting
information. A general trend of redꢀshift of the absorption band maximum was observed from azaꢀ
BODIPYs A1, A2 to A4 in the calculated absorption spectra (Fig. S2). For example, azaꢀBODIPY A2
shows a 49 nm redꢀshift in absorption with respect to that of azaꢀBODIPY A1, which is close to the
experimental result. More importantly, azaꢀBODIPY A4 bearing electronꢀwithdrawing cyano groups
exhibits a 40 nm further redꢀshift of the absorption with respect to that of azaꢀBODIPY A2. The
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calculation of azaꢀBODIPY A4 also gives an oscillator strength of 0.7946 for the S0 S1 transition. The
→
1
2
3
4
5
6
7
8
9
comparison of the HOMO and LUMO orbital energies clearly demonstrated that the methoxy and cyano
groups have different impact on the absorptions of azaꢀBODIPYs. For azaꢀBODIPY A2 containing only
methoxy groups at para-positions of 3,5ꢀphenyls, more increases of HOMO than LUMO energy was
observed, which leads to the decrease of excitation energy and a redꢀshift of the absorption band in azaꢀ
BODIPY A2. While in azaꢀBODIPY A4 with a further installation of cyano groups on the paraꢀ
positions of 1,7ꢀphenyls with respect to A2, not only the increase of HOMO energy but also the
decreases of LUMO energy were observed. In another word, the installation of both the methoxy on the
para-positions of 3,5ꢀphenyls and the cyano groups on the para-positions of 1,7ꢀphenyls facilitates the
reduce of the excitation energy in azaꢀBODIPY A4.
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Encouraged by these DFT calculations, we synthesized azaꢀBODIPYs A1ꢀA5 in three steps from the
aldol condensation of aldehydes and ketones, Michael addition of nitromethane, followed by
condensation with ammonium acetate and the subsequent BF3 complexation using reported methods5a
(Scheme 1). A6 was synthesized in 81% yield from the reaction of A5 with methyl iodide. All these new
compounds were characterized by NMR and HRMS.
Photophysical properties of azaꢀBODIPYs A3ꢀA6 in solvents with different polarities were studied as
shown in Fig. 2 and summarized in Table 2. For comparison purposes, the reference compounds A1 and
A2 were also evaluated in three different solvents. In comparison with those of A2, our azaꢀBODIPYs
A3, A4 and A6 generally exhibit 15ꢀ28 nm and 17ꢀ30 nm bathochromic shifts for absorption and
emission, respectively. In the various solvents studied, azaꢀBODIPY A4 bearing two cyano groups (σp =
0.66) at the paraꢀpositions of 1,7ꢀphenyls shows the longest λmax for both absorption (720 nm) and
emission (756 nm). Interestingly, in agreement with the DFT calculations, these dyes all show a much
enhanced absorption at about 470 nm in comparison with those of A1 and A2. This band can be
assigned to the S0 S3 transition according to the DFT calculations and can be used to excite these dyes
→
to achieve larger Stokes shifts. This type of transition has previously been reported in several distyrylꢀ
BODIPY dyes and indicates the existence of a pushꢀpull effect in those dyes.13,15
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Table 2. Photophysical properties of azaꢀBODIPYs in different solvents.
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3
4
5
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SS (cmꢀ1) b
672
λ
abs (nm)
λemsa (nm)
684
φfac
Dyes
solvent
toluene
654
650
645
693
688
685
709
704
709
700
697
720
716
718
710
706
0.44
0.34
0.17
0.39
0.36
0.26
0.38
0.34
0.28
0.21
0.26
0.36
0.29
0.22
0.16
0.21
7
8
9
A1
chloroform
methanol
toluene
682
722
673
645
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723
599
A2
A3
chloroform
methanol
toluene
723
704
720
710
740
591
chloroform
THF
741
709
744
664
methanol
acetonitrile
toluene
739
754
740
834
754
626
chloroform
THF
755
721
756
700
A4
methanol
acetonitrile
toluene
750
751
750
831
not soluble
chloroform
THF
712
714
706
702
752
750
743
744
747
672
705
804
0.14
0.16
0.14
0.18
A6
methanol
acetonitrile
aA1 excited at 610 nm, A2-A6 excited at 670 nm. bSS: Stokes shift. the fluorescence quantum yields of
A2-A6 were calculated using A2 in CHCl3 (φ = 0.36) as the standard.
c
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1.0
(b)
1.0
0.5
0.0
(a)
1
2
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8
0.5
9
10
11
12
13
14
0.0
650
700
750
800
850
300
400
500
600
700
800
Wavelength (nm)
Wavelength (nm)
Figure 2. Normalized absorption (a) and fluorescence spectra (b) of azaꢀBODIPYs A1 (black), A2 (red),
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A3 (green), A4 (blue) and A6 (orange) in CHCl3.
AzaꢀBODIPYs A3, A4 and A6 exhibit moderate fluorescent quantum yields (0.14ꢀ0.38) in various
solvents studied, with respect to that of A2. These dyes show a relative longer wavelength absorption
and emission with higher fluorescence quantum yields in nonpolar solvents, similar to those of the
reference compound A2. The solvent dependence of photophysical properties are also in good
agreement with classic BODIPYs containing electronꢀdonating groups as disclosed in detail by Boens.16
To demonstrate the possible utility of our azaꢀBODIPY dyes, compounds A3, A4 and A6 were applied
for cell imaging. They were incubated at the concentration of 10 ꢁM for 6 h with human carcinoma
HEp2 cells at 37 °C. All azaꢀBODIPYs were found to rapidly accumulate within the cells and gave
bright red fluorescence, as shown in Fig. 3b and Fig. S3b in the supporting information. To investigate
the main intracellular sites of the localization, the HEp2 cells were coꢀincubated with each azaꢀBODIPY
and ER Tracker Blue/White (ER) at 100 nM for 30 min, MitoTracker Green (mitochondria) at 250 nM
for 30 min, BODIPY FL C5ꢀceramide (Golgi) at 50 nM for 30 min and LysoSensor Green (lysosomes)
at 50 nM for 30 min. The corresponding overlay images were shown in Figs. 3d, 3f, 3h and 3j. These
results indicate that azaꢀBODIPY A4 localizes in all the subcellular sites tested, while A3 and A6
localize preferentially in the cell mitochondria, lysosomes and Golgi apparatus. We also studied the
phototoxicity of this series of azaꢀBODIPYs, since high cytotoxicity would limit their potential
applicability in cellular imaging. The cytotoxicity of A3, A4 and A6 was evaluated using the Cell Titer
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Blue viability assay, at concentrations up to 100 ꢀM, upon exposure to 1 J/cm2 light dose (see Fig. S4 in
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the supporting information). None of the dyes showed any phototoxicity up to 100 ꢀM concentrations.
These results are in agreement with previous studies,17ꢀ18 and warrant further investigation of these dyes
as bioimaging probes.
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Figure 3. Subcellular localization of azaꢀBODIPYs A3 (left) and A4 (right) in HEp2 cells at 10 ꢁM for
6 h. (a) Phase contrast, (b) overlay of A3/A4 fluorescence and phase contrast, (c) ER tracker Blue/White
fluorescence, (e) MitoTracker Green fluorescence, (g) BODIPY Ceramide fluorescence, (i) LysoSensor
Green fluorescence, and (d, f, h, j) overlays of organelle tracers with A3/A4 fluorescence. Scale bar: 10
ꢀm.
In summary, the installation of strong electronꢀwithdrawing groups on the paraꢀpositions of 1,7ꢀ
phenyls in azaꢀBODIPYs resulted in novel long wavelength NIR azaꢀBODIPYs that emit above 740 nm,
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are highly cell permeable and show very low cytotoxicity. This strategy provides a simple approach to
the development of redꢀshifted azaꢀBODIPY dyes, through a pushꢀpull effect.
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Experimental Section
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General. The NMR experiments were obtained on a 300 MHz NMR spectrometer at room temperature.
Chemical shifts (δ) are given in ppm relative to TMS. Highꢀresolution mass spectra were obtained using
APCIꢀTOF in positive mode. UVꢀvisible absorption spectra and fluorescence emission spectra were
recorded on a commercial spectrophotometer (190ꢀ900 nm scan range). The slit width was set at 2.5 nm
for excitation and 5.0 nm for emission. Relative fluorescence quantum yields were calculated using A2
in CHCl3 (φ = 0.36) as the standard. All φ values are corrected for changes in refractive index using a
previous reported method.19
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Computational Details. The ground state geometries of molecules A1, A2 and A4 were fully optimized
with DFT method under B3LYP/6ꢀ31g level. The stationary structures have been verified with no
imaginary vibration in the frequency calculations. The vertical excitation properties have been estimated
by taking TDꢀDFT singleꢀpoint calculations under the same level with the optimized ground state
geometries. The solvation by chloroform has been estimated in the calculations under the PCM scheme
as well. All of the calculations were carried out by the methods implemented in Gaussian 09 package.20
Synthesis of 1a. To 4ꢀ(trifluoromethyl)benzaldehyde (3.0 mL, 0.02 mol) and 4ꢀmethoxyacetophone (3.3
g, 0.02 mol) in anhydrous methanol (20 mL) were added 3 g of KOH. The mixture was stirred at room
temperature for 1 h. The precipitate was filtered, washed with methanol and dried under reduced
pressure to give 1a as a light yellow solid in 86% yield (5.5 g). 1H NMR (CDCl3, 300 MHz) δ: 8.07 (d, J
13
= 9.0 Hz, 2H), 7.59ꢀ7.84 (m, 6H), 7.01 (d, J = 6 Hz, 2H), 3.91 (s, 3H). C NMR (CDCl3, 75 MHz) δ:
188.2, 163.7, 141.9, 138.5, 131.5, 130.9, 130.7, 128.4, 125.9 (q, J = 15 Hz), 124.0, 122.1, 114.0, 55.6.
HRMS (APCI) calcd. for C17H13F3O2 [M+H]+: 307.0940, found 307.0940. mp 161 ꢀ 163 oC.
1b was synthesized as a light yellow solid in 92% yield (4.8 g) using the above procedure from 4ꢀ
cyanobenzaldehyde (2.6 g, 0.02 mol) and 4ꢀmethoxyacetophone (3.0 g, 0.02 mol). 1H NMR (CDCl3, 300
13
MHz) δ: 8.03 (d, J = 9.0 Hz, 2H), 7.57ꢀ7.69 (m, 6H), 6.98 (d, J = 9.0 Hz, 2H), 3.88 (s, 3H). C NMR
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(CDCl3, 75 MHz) δ: 187.9, 163.8, 141.3, 139.4, 132.7, 131.0, 130.5, 128.6, 125.0, 118.5, 114.0, 113.2,
55.6. HRMS (APCI) calcd. for C17H13NO2 [M+H]+: 264.1019, found 264.1017. mp 171 ꢀ 172 oC.
1c was synthesized as a light yellow solid in 85% yield (4.8 g) using the above procedure from 4ꢀ
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dimethylaminobenzaldehyde (3.0 mL, 0.02 mol) and 4ꢀmethoxyacetophone (3.0 g, 0.02 mol). H NMR
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(CDCl3, 300 MHz) δ: 8.04 (d, J = 6.0 Hz, 2H), 7.80 (d, J = 18.0 Hz, 2H), 7.56 (d, J = 6.0 Hz, 2H), 7.37
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(d, J = 15.0 Hz, 2H), 6.98 (d, J = 6.0 Hz, 2H), 6.71 (d, J = 9.0 Hz, 2H), 3.89 (s, 3H), 3.05 (s, 6H). C
NMR (CDCl3, 75 MHz) δ: 188.9, 163.0, 151.9, 145.0, 131.9, 130.6, 130.3, 122.8, 116.6, 113.7, 111.8,
55.5, 40.2. HRMS (APCI) calcd. for C17H13NO2 [M+H]+: 282.1489, found 282.1482. mp 141 ꢀ 143 oC.
Synthesis of 2a. To compound 1a (6.1 g, 20 mmol) in anhydrous methanol (30 mL) were added
diethylamine (10 mL) and nitromethane (10 mL). The mixture was refluxed for 10 h, concentrated under
vacuum to afford 2a in 97% yield (7.1 g). 1H NMR (CDCl3, 300 MHz) δ: 7.87 (d, J = 9.0 Hz, 2H), 7.53
(d, J = 9.0 Hz, 2H), 7.43 (d, J = 9.0 Hz, 2H), 6.89 (d, J = 9.0 Hz, 2H), 4.83ꢀ4.89 (m, 1H), 4.66ꢀ4.73 (m,
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1H), 4.27ꢀ4.31 (m, 1H), 3.79 (s, 3H), 3.39ꢀ3.41 (m, 2H). C NMR (CDCl3, 75 MHz) δ: 193.9, 162.9,
142.7, 129.3, 128.7, 128.1, 127.1, 124.8 (q, J = 15 Hz), 117.6, 112.4, 78.1, 54.4, 39.8, 38.1. HRMS
(APCI) calcd. for C18H16F3NO4 [M+H]+: 368.1140, found 368.1107. mp 141 ꢀ 142 oC.
2b was synthesized as a yellow oily product in 96% yield (3.1 g) using the above procedure from 1b (2.6
g, 10 mmol). 1H NMR (CDCl3, 300 MHz) δ: 7.85 (d, J = 6.0 Hz, 2H), 7.59 (d, J = 9.0 Hz, 2H), 741 (d, J
= 9.0 Hz, 2H), 6.89 (d, J = 6.0 Hz, 2H), 4.80ꢀ4.87 (m, 1H), 4.64ꢀ4.71 (m, 1H), 4.23ꢀ4.28 (m, 1H), 3.83
(s, 3H), 3.37 (d, J = 6.0 Hz, 2H). 13C NMR(CDCl3, 75 MHz) δ: 194.6, 164.0, 144.9, 132.7, 130.3, 129.0,
128.6, 118.5, 114.0, 111.6, 78.9, 55.6, 40.6, 39.3. HRMS (APCI) calcd. For C18H16N2O4 [M+H]+:
325.1183, found 325.1181. mp 93 ꢀ 94 oC.
2c was synthesized as a yellow oily product in 89% yield (6.1 g) using the above procedure from 1c (5.6
g, 20 mmol). 1H NMR (CDCl3, 300 MHz) δ: 7.91 (d, J = 6.0 Hz, 2H), 7.14 (d, J = 9.0 Hz, 2H), 6.93 (d,
J = 9.0 Hz, 2H), 6.68 (d, J = 9.0 Hz, 2H), 4.76ꢀ4.82 (m, 1H), 4.59ꢀ4.66 (m, 1H), 4.11 (m, 1H), 3.87 (s,
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3H), 3.35 (d, J = 6.0 Hz, 2H), 2.92 (s, 6H). C NMR (CDCl3, 75 MHz) δ: 194.8, 162.7, 148.9, 129.3,
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128.5, 127.0, 125.5, 112.8, 111.7, 79.0, 54.5, 41.3, 40.4, 39.4. HRMS (APCI) calcd. for C19H22N2O4
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[M+H]+: 343.1652, found 343.1643. mp 113 ꢀ 114 oC.
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Synthesis of A3. A mixture of 2a (0.37 g, 1.0 mmol) and ammonium acetate (1.5 g, 20 mmol) in
ethanol (20 mL) were refluxed for 9 h, cooled down to room temperature and concentrated to about 5
mL. The precipitate was filtered, washed with water and ethanol to give the intermediate azaꢀ
dipyrromethene as a metallic blueꢀblack powder. 1H NMR (CDCl3/CF3COOH, 300 MHz) δ: 11.87 (brs,
2H), 7.95 (d, J = 8.1 Hz, 4H), 7.62 (d, J = 7.8 Hz, 4H), 7.49 (d, J = 7.8 Hz, 4H), 7.30 (s, 2H), 7.06 (d, J
= 8.4 Hz, 4H), 3.94 (s, 6H). This azaꢀdipyrromethene was directly used for the subsequent BF3
complexation reaction without further purification: to azaꢀdipyrromethene (0.15 g, 0.23 mmol) in
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toluene (100 mL) was added triethylamine (4 mL) and BF3 OEt2 (6 mL). The mixture was stirred at 60
oC for 2hand removed solvents under vacuum. The residue was washed with ethanol, and further
purified by recrystallization from dichoromethane/methanol or by passing a small plug of silica gel
using dichloromethane/hexane as eluent to give the product as a red copperꢀcolored solid in 46% yield
over two steps (0.14 g). 1H NMR (CDCl3, 500 MHz) δ: 8.09 (d, J = 10.0 Hz, 4H), 8.08 (d, J = 10.0 Hz,
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4H), 7.68 (d, J = 10.0 Hz, 4H), 7.09 (s, 2H), 7.00 (d, J = 10.0 Hz, 4H), 3.88 (s, 6H). C NMR (CDCl3,
125 MHz) δ: 162.3, 159.0, 145.2, 141.4, 135.6, 131.8, 130.8 (q, JF-C = 31.3 HZ), 129.1, 125.5 (q, JF-C
=
2.5 HZ), 124.1 (q, JF-C = 270 HZ), 123.9, 119.8, 114.5, 55.4. HRMS (APCI) calcd. for C36H24BF8N3O2
[M+H]+: 694.1907, found 694.1896. mp﹥260 oC.
A4 was synthesized using the above procedure from 2b (0.37 g, 1.0 mmol) and ammonium acetate (1.5
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g, 20 mmol). The intermediate azaꢀdipyrromethene was collected as black powder. H NMR
(CDCl3/CF3COOH, 300 MHz) δ: 11.94 (brs, 2H), 8.03 (d, J = 8.1 Hz, 4H), 7.80 (d, J = 7.8 Hz, 4H),
7.67 (d, J = 7.8 Hz, 4H), 7.38 (s, 2H), 7.11 (d, J = 8.1 Hz, 4H), 3.96 (s, 6H). It was directly used for the
subsequent BF3 complexation without further purification to afford azaꢀBODIPY A4 as a greenish solid
in 37 % yields over two steps (0.13 g). 1H NMR (CDCl3, 300 MHz) δ: 8.09 (m, 8H), 7.74 (d, J = 6.0 Hz,
4H), 7.13 (s, 2H), 7.04 (d, J = 9.0Hz, 4H), 3.91 (s, 6H). 13C NMR was not available due to poor
solubility. HRMS (APCI) calcd. for C36H25BF2N5O2 [M+H]+: 608.2064, found 608.2035. Elemental
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analysis calcd. (%) for C36H24BF2N5O2: C 71.18, H, 3.98, N 11.53; found: C 70.89, H 3.77, N 11.27. mp
﹥260 oC.
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A5 was synthesized using the above procedure from 2c (0.34 g, 1.0 mmol) and ammonium acetate (1.5
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g, 20 mmol). The intermediate azaꢀdipyrromethene was collected as a black powder. H NMR (CDCl3,
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300 MHz) δ: 8.00 (d, J = 8.1 Hz, 4H), 7.84 (d, J = 8.1 Hz, 4H), 7.00ꢀ6.95 (m, 6H), 7.38 (s, 2H), 6.74 (d,
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J = 8.1 Hz, 4H), 3.86 (s, 6H), 2.99 (s, 12H). C NMR (CDCl3, 75 MHz) δ: 160.6, 153.7, 150.1, 149.1,
142.0, 129.9, 127.9, 125.6, 122.7, 114.4, 112.0, 111.2, 55.4, 40.4. It was directly used for the BF3
complexation without further purification to afford AzaꢀBODIPY A5 as a greenish solid in 44% yield
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over two steps (0.14 g). H NMR (CDCl3, 500 MHz) δ: 8.08 (d, J = 5.0 Hz, 4H), 8.05 (d, J = 10.0 Hz,
4H), 6.98 (d, J = 10.0 Hz, 4H), 6.83 (brs, 6H), 3.87 (s, 6H), 3.09(s, 12H). 13C NMR (CDCl3, 125 MHz)
δ: 161.3, 156.9, 150.8, 145.3, 142.9, 131.2, 130.8, 125.0, 121.3, 115.1, 114.0, 112.2, 55.6, 40.4. HRMS
(APCI) calcd. for C38H36BF2N5O2 [M+H]+: 644.3003, found 644.2976. mp﹥260 oC.
Synthesis of A6. AzaꢀBODIPY A5 (0.13 g, 0.2 mmol) and methyl iodide (3.7 mL, 60 mmol) were
o
stirred in dry chloroform (20 mL) at 50 C in dark for 48 h. The precipitate was filtered, washed with
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chloroform and dried under vacuum to give azaꢀBODIPY A6 as a brown solid in 81% yield (0.15 g). H
NMR (DMSOꢀd6, 300 MHz) δ: 8.37 (d, J = 9.0Hz, 4H), 8.21 (m, 8H), 7.76 (s, 2H), 7.14 (d, J = 9.0 Hz,
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4H), 3.90 (s, 6H), 3.24 (s, 18H). C NMR (DMSOꢀd6, 75 MHz) δ: 162.8, 158.2, 147.9, 145.2, 140.2,
133.7, 132.5, 130.9, 123.3, 121.5, 121.4, 115.1, 57.0, 56.2. HRMS (APCI) calcd. for C38H36BF2N5O2
[Mꢀ2CH3+H]+: 644.3003, found 644.2980. Elemental analysis calcd. (%) for C40H42BF2I2N5O2: C 51.80,
H, 4.56, N 7.55; found: C 51.44, H 4.19, N 7.20. mp﹥260 oC.
Cell Culture: All tissue culture media and reagents were obtained from Invitrogen. Human HEp2 cells
were obtained from ATCC and maintained in a 50:50 mixture of DMEM: Advanced MEM containing
5% FBS, 1% Primocin antibiotic in a humidified, 5% CO2 incubator at 37 °C. The cells were
subcultured biweekly to maintain subconfluent stocks. The compounds were dissolved in DMSO and
1% of Cremophor to make a 400 ꢀM stock solution.
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Microscopy: The cells were incubated in a glass bottom 6ꢀwell plate (MatTek) and allowed to grow for
48 h. The cells were then exposed to 10 ꢁM of each compound for 6 h. Organelle tracers were obtained
from Invitrogen and used at the following concentrations: LysoSensor Green 50 nM, MitoTracker Green
250 nM, ER Tracker Blue/white 100 nM, and BODIPY FL C5 ceramide 1 mM. After 30 min incubation
in the 37 °C, 5% CO2 incubator, both the media and the organelle tracers were removed and washed
with PBS buffer for 3 times. Images were acquired using a Leica DMRXA microscope with 40× NA 0.8
dip objective lens and DAPI, GFP, and Texas Red filter cubes (Chroma Technologies).
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Phototoxicity: HEp2 cells were plated at 10 000 per well in a Costar 96 well plate and allowed 48 h to
attach. The cells were exposed to increasing concentrations of azaꢀBODIPYs to 100 ꢂM. After
compound loading overnight, the medium was removed and replaced with medium containing 50 mM
HEPES pH 7.2. The cells were then placed on ice and exposed to light from a 100 W halogen lamp
filtered through a 610 nm long pass filter (Chroma) for 20 min. An inverted plate lid filled with water to
a depth of 5 mm acted as an IR filter. The total light dose was approximately 1 J/cm2. The cells were
returned to the incubator for 24 h. The loading medium was then removed and the cells fed medium
containing Cell Titer Blue (Promega) as per manufacturer’s instructions. Cell toxicity was then
measured by reading the fluorescence at 520/584 nm using a BMG FLUOstar plate reader. The signal
was normalized to 100% viable (untreated) cells and 0% viable (treated with 0.2% saponin from Sigma)
cells.
Acknowledgement. This work is supported by the National Nature Science Foundation of China
(Grants No. 21072005, 21272007 and 21372011), National Science Foundation of Anhui Province
(1208085MB29) and the United States National Science Foundation (CHEꢀ0911629).
Supporting Information Available: Copies of NMR spectra and high resolution mass spectra for all new
compounds, calculated frontier orbitals and absorption spectra, subcellular localization of azaꢀBODIPY
A6 and cytotoxicities of azaꢀBODIPYs A3, A4 and A6 are available free of charge via the Internet at
http://pubs.acs.org.
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