Benzimidazole-BODIPY as optical and fluorometric pH sensor

Article abstract of DOI:10.1016/j.dyepig.2016.01.029

A new optical and fluorometric pH sensor has been obtained by attaching pH sensitive group benzimidazole at the 2-position of borondipyrromethene. Due to the electron-donor effect, benzimidazole group caused obvious bathochromic shift in the absorption an

Full text of DOI:10.1016/j.dyepig.2016.01.029

Dyes and Pigments 128 (2016) 165e169
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Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
Benzimidazole-BODIPY as optical and fluorometric pH sensor
a
b
a
a
a, *
Zhensheng Li , Lei-Jiao Li , Tingting Sun , Liming Liu , Zhigang Xie
a
State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 5625 Renmin Street,
Changchun 130022, PR China
State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022,
b
PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 November 2015
Received in revised form
A new optical and fluorometric pH sensor has been obtained by attaching pH sensitive group benz-
imidazole at the 2-position of borondipyrromethene. Due to the electron-donor effect, benzimidazole
group caused obvious bathochromic shift in the absorption and fluorescence spectra. Upon protonation,
the solution exhibited drastic blue-shifted absorption and enhanced fluorescence, and the color changed
from yellow to green simultaneously. The sensing mechanism was elucidated by quantum chemistry
calculation approach. Cell experiments were carried out to verify the compound could selectively locate
in the acidic lysosome. These results indicated the Benzimidazole-BODIPY could be used as an optical and
27 January 2016
Accepted 29 January 2016
Available online 6 February 2016
Keywords:
“off-on” fluorescent pH indicator.
Borondipyrromethene
Benzimidazole
Lysosome labeling
pH probe
©
2016 Elsevier Ltd. All rights reserved.
1. Introduction
modify the fluorophore to develop intracellular pH sensors [18,19].
For example, G o€ khan Sevinç et al. have reported 8-substituted
The precise pH measurement attracts much attention of re-
benzimidazole-BODIPY pH sensor [20]. Unfortunately, the intense
fluorescence was quenched drastically with addition of H . More-
over the protonation could not cause a substantial absorption
change due to the orthogonal conformation.
þ
searchers for their potential application in environmental moni-
toring and biological systems. Among different analytic methods,
fluorescent-based analyses are promising tools due to low cost,
convenient operations, high sensitivity, excellent spatial and tem-
poral resolution [1e4]. Although much work about pH sensor was
reported, it remains challenging to design and synthesize novel
fluorescent pH sensors with high sensitivity [5e10].
Herein, we synthesized a novel 2-substituted BODIPY (BDP-BIM)
which could be used as an optical and “turn-on” fluorescent pH
sensor. The compound exhibited intense fluorescence in nonpolar
solvents but not in polar solvents, while the photophysical spectra
did not change apparently. On the other hand, the absorption and
emission peaks of BDP-BIM showed obvious blue shift upon pro-
tonation of benzimidazole in aqueous solution with greatly
increased fluorescence. The distinct spectra properties could be
explained by electron effect as confirmed by density functional
theory (DFT) calculation. It is expected that the designed compound
can be used a dual pH and polarity probe due to 2-substituted
benzimidazole moiety. Furthermore, the probe could respond to
acid environment inside the cells and selectively label the lysosome.
Borondipyrromethene
(BODIPY)
fluorophores
possess
outstanding photophysical properties, including high molar ab-
sorption coefficients and fluorescence quantum yields, narrow
emission bandwidths, and high stability in physiological conditions
[
11,12]. Moreover, their spectroscopic characteristics can be fine-
tuned by introducing appropriate substituents at different posi-
tion of the BODIPY backbone. These unique advantages facilitate
the design various fluorescent probes based on BODIPY in analyte-
detection, bio-imaging and labeling [13e17]. Benzimidazole group
has two acidebase equilibrium with pK
which is ideally located within the physiological range of acidic
a
¼ 5.7, 12.6, the former of
2. Experiment section
organelles, such as lysosome (pH 4.5e5.5) and endosome (pH
4
.5e6.8). So benzimidazole derivatives are usually employed to
2.1. General information
Solvents and reagents were purchased commercially as reagent
grade and used as received unless otherwise mentioned.
*
Corresponding author. Tel.: þ86 431 85262775.
E-mail address: xiez@ciac.ac.cn (Z. Xie).
http://dx.doi.org/10.1016/j.dyepig.2016.01.029
143-7208/© 2016 Elsevier Ltd. All rights reserved.
0

166
Z. Li et al. / Dyes and Pigments 128 (2016) 165e169
ꢁ
Dichloromethane and N, N-dimethylformamide (DMF) were
distilled over calcium hydride. The solvents used for spectroscopic
Then the solution was heated and stirred at 80 C for 4 h. After
cooling to room temperature, the reaction solution was quenched
measurements were of HPLC grade.
pared according to the reported procedure [21]. The pH sensing
experiment was carried in the CH OH/H O (1:1 v/v) solution. All of
the quantum calculations were performed using the Gaussian09
program package [22]. The ground state structure was optimized
using the density functional theory (DFT) method with the hybrid-
generalized gradient approximation (H-GGA) functional B3LYP.
b
-Formyl-BODIPY was pre-
2 3
by addition of Na CO aqueous (0.1 mmol, 20 mL), then the mixture
was extracted with EtOAc (2 ꢀ 20 mL). The organic phase was dried
over anhydrous sodium sulfate. Evaporation of solvent gave the
crude product, which was purified by column chromatography over
silica gel (hexane/ethyl acetate, 3/1) to afford the red solid product
3
2
1
6
(yield: 82%). H NMR (400 MHz, DMSO-d )
d 12.29 (s, 1H), 7.61 (d,
J ¼ 6.5 Hz, 4H), 7.46 (d, J ¼ 7.6 Hz, 3H), 7.18 (s, 2H), 6.32 (s, 1H), 2.65
1
3
(
(
s, 3H), 2.52 (d, J ¼ 5.1 Hz, 3H), 1.50 (s, 3H), 1.39 (s, 3H). C NMR
6
101 MHz, DMSO-d )
d 157.86, 152.88, 146.12, 144.84,142.86, 139.57,
2.2. Instruments
133.89, 131.92, 129.77, 129.55, 129.46, 127.78, 122.76, 122.45, 121.76,
1H NMR and 13C NMR spectra were recorded on a Bruker NMR
00 DRX Spectrometer using DMSO-d as solvent. Electrospray
14.47, 14.21, 13.43, 12.50. ESI-MS calculated ([C
z ¼ 440.2, found m/z ¼ 439.3 [M ꢂ H] .
26H23BF N ]) m/
2
4
ꢂ
6
4
Ionization (ESI) mass spectra were obtained on a Finnigan LCQ
quadrupole ion trap mass spectrometer. UVeVis absorption and
emission spectra were obtained using a Shimadzu UV-2450 PC
UVeVis Spectrophotometer and a Hitachi F-4500 Fluorescence
Spectrophotometer, respectively. The luminescence quantum
yields in solution were measured by using rhodamine 6G with
3. Result and discussion
3.1. Synthesis and characterization
Most fluorescent BODIPY-based sensors are modified on
meso position [24,25]. -Position functionalization strategies are
less investigated. Formylation of the -position of BODIPY via
a or
excitation wavelength 488 nm (
F
F
¼ 0.86 in MeOH) as reference
b
[23].
b
VilsmeiereHaack reaction provides versatile functionalization ap-
proaches. BDP-BIM was synthesized by the facile route shown in
Scheme 1. The electron-deficient BODIPY system facilitates the
condensation of aldehyde group and benzene-1,2-diamine to
obtain product in high yield. The compound was characterized by
2
.3. Confocal laser scanning microscopy (CLSM)
BDP-BIM was dissolved in DMSO with a concentration of
1.0 mM, and diluted to 5 mM in phosphate-buffered saline (PBS)
1
13
solution. Cellular uptake by human cervical carcinoma (HeLa) cells
was examined with CLSM. HeLa cells were seeded in 6-well culture
plates (a clean cover slip was put in each well) at a density of
H NMR, C NMR, ESI-MS analysis and spectroscopic methods. The
1
sharp signal at 12.29 ppm in the H NMR spectrum (Fig. S1) was
assigned to the active hydrogen of imidazole ring.
4
5
ꢀ 10 cells per well and allowed to adhere for 24 h. The medium
was then replaced by BDP-BIM in PBS solution (containing 0.5%
DMSO). After incubation for 0.5 h at 37 C, the supernatant was
3.2. Spectroscopic properties
ꢁ
carefully removed and the cells were washed three times with PBS.
The spectra properties of BDP-BIM in various solvents were
recorded as summarized in Table 1. As shown in Fig. 1, the ab-
sorption spectra inherit characteristics of typical BODIPY dyes, and
Subsequently, Lyso-Tracker Red was used to stain lysosomes for
ꢁ
4
5 min at 37 C. After being washed with PBS, the cells were fixed
with 1 mL of 4% formaldehyde each well for 10 min at room tem-
perature and washed twice with PBS again. Samples were exam-
ined by CLSM using a Zeiss LSM 700 (Zurich, Switzerland).
the intense S
benzimidazole substituent, the shoulder peak ascribed to the 0e1
vibration band of S eS transition attenuates significantly, mean-
0 1
eS transition centers around 510 nm. Due to the 2-
0
1
while the maximum peaks display red shifts of 10e20 nm
compared to that of unsubstituted BODIPY. The emission spectra
exhibit similar red-shifted behaviors. These results confirm the
“pushepull” electron interaction between the benzimidazole
fragment and BODIPY core. Although the main absorption and
2
.4. Synthetic procedures
b-Formyl-BODIPY (0.5 mmol), o-phenylenediamine (0.5 mmol)
and p-TsOH (0.1 mmol) were thoroughly mixed in DMF (10 mL).
Scheme 1. The synthetic pathway of compound BDP-BIM.

Z. Li et al. / Dyes and Pigments 128 (2016) 165e169
167
Table 1
Spectroscopic data of BDP-BIM in various solvents.
max (dm3 mol cm
ꢂ1
ꢂ1
)
lem/nm
Stokes shift/nm
F
F
Compound
Media
labs/nm
ε
BDP-BIM
Hexane
Toluene
512
515
510
511
513
513
506
508
99,000
82,600
563
564
562
558
568
571
548
565
51
49
52
47
55
58
42
57
0.57
0.52
0.21
0.34
0.18
0.05
0.10
0.07
CH
2
Cl
2
102,000
73,900
80,100
80,400
86,600
84,000
CHCl
THF
3
DMF
CH
CH
3
OH
CN
3
Fig. 1. Absorption (a) and fluorescence (b) spectra of BDP-BIM in various solvents.
þ
Fig. 2. The changes of absorption (a) and emission (b) spectra in the CH
emission at 520 nm; (d) fluorescence enhancement effect (F and F
3
OH/H
2
O (1:1 v/v) solution upon addition of H ; (c) the sigmoidal fitting of absorbance at 505 nm and
0
are emission intensity at 520 nm in different pH intervals and at 560 nm in neutral condition, respectively).

168
Z. Li et al. / Dyes and Pigments 128 (2016) 165e169
therefore we added base to the neutral solution to examine the
ꢂ
OH response. Although the fluorescence of BDP-BIM was
enhanced below pH 3, slightly hyperchromic effect and fluores-
cence quenching were observed with increasing alkaline concen-
tration. These findings indicate that the improved emission
intensity is related to the photoinduced electron transfer (PET)
mechanism between the benzimidazole and BODIPY groups.
3.4. Density functional theory (DFT) calculation
In order to prove our speculation, theoretical calculations are
performed with density functional theory by using Gaussian09
software package. The structures of the neutral and protonated BDP-
BIM are optimized at the B3LYP/6-31 þ G(d) level [29,30], followed
by the analysis on the frontier orbitals. As shown in Fig. 3, HOMO of
neutral BDP-BIM was localized on the BODIPY core and benzimid-
azole, while LUMO was mainly contributed by the BODIPY units.
However, both HOMO and LUMO of protonated BDP-BIM were
donated by the orbital from the BODIPY core, demonstrating that
photoinduced electron transfer partly existed between BODIPY and
benzimidazole moiety in neutral form, whereas this electron
transfer was inhibited in protonated form. The recognition mecha-
nism of this probe was completely opposite to that of 8-substituted
analogy reported by Sevinç [20], resulting in our “light-up” probe.
The energy band-gap was calculated to be 2.84 eV for neutral form
and 3.12 eV for protonated form, reflecting increased transition
energy, which was in well accordance with the blue-shift spectra.
Fig. 3. Molecular orbital energy levels of BDP-BIM in neutral and protonated form.
emission show little solvent dependence, the fluorescence emis-
sion quantum yields vary dramatically in different solvent. For
example, the
be 0.57, whereas it is 0.07 in CH
F
F
in nonpolar solvent such as hexane is measured to
CN. This polarity dependence is
3
probably due to the positive solvatokinetic effect [26e28], facili-
tating “off-on” probing in polar media. In addition, the large
hypochromatic-shifted spectra in CH OH are ascribed to the po-
3
larity and protonation dual effect, which can be verified by the
following pH titration experiment.
3.5. Intracellular pH response
a
The probe with pK value of 5.2 is expected to response to acidic
3.3. pH-dependent absorption and fluorescence spectra
media in the living cells. To assess whether BDP-BIM can be utilized
as an organelle probe, the HeLa cells were labeled with our probe
and Lyso-Tracker Red. The bright fluorescence was observed in the
cells (Fig. 4a), indicating the efficient cell uptake and intracellular
protonation of BDP-BIM. As shown in Fig. 4c, the green fluor-
escence of BDP-BIM can superimpose with red fluorescence (in the
web version) of Lyso-Tracker Red mostly. This result confirmed that
our probe could track acidic lysosome in the cells as well. The
ability to sense acidic and near neutral pH in the cell using fluo-
rescent probe could facilitate biological study associated with pH
change in lysosome and cytosol.
The spectrophotometric pH titrations were carried out to
examine the spectra changes. As shown in Fig. 2a, when pH value
decreased from 8.09 to 3.05, the absorption at 505 nm declined.
While a new peak at 490 nm increased gradually with the appear-
ance of isobestic points at 498 and 409 nm. The 15 nm blue-shifted
absorption attributed to the inhibition electron effect of benzimid-
azole. The colorof solution changed fromlight yellowtobright green
þ
(
in the web version), indicating the ability of optical detection of H
ion. The emission spectra changes depicted the same proto-
nationedeprotonation process. At first, the BDP-BIM solution
emitted weak fluorescence at 560 nm. With continuous addition of
acid, the yellow emission vanished, and the new band at 520 nm
4. Conclusion
increased significantly with a large fluorescent “off-on” ratio of 26
*
(
Fig. 2d). The pK
a
and pK
a
values were determined to be 5.2 and 4.8
In summary, we have developed an optical and “off-on” fluo-
rescent pH sensor BDP-BIM. Upon protonation, the solution color
changed from yellow to green with a large fluorescence “off-on”
ratio of 26. The probing mechanism was different from the 8-
by the nonlinear sigmoidal fitting of titration data, respectively.
Benzimidazole possessed two acidebase equilibriums of pK
values of 5.7 (protonated-neutral) and 12.6 (neutral-anionic),
a
Fig. 4. Confocal laser scanning images of HeLa cells co-labeled with BDP-BIM (a) in PBS solution containing 0.5% DMSO and Lyso-Tracker Red (b), (c) merged image from (a) and (b).
Scale bar ¼ 20 m.
m

Z. Li et al. / Dyes and Pigments 128 (2016) 165e169
169
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