A charge transfer type pH responsive fluorescent probe and its intracellular application

Article abstract of DOI:10.1039/b9nj00703b

A new charge transfer pH fluorescent probe BTP has been prepared by the ethylene bridging of benzothiazole and pyridine. The probe exhibits a specific fluorescent response to pH with a large Stokes shift, and the pH-induced emission enhancement factor (EEF) is about 22-fold when the pH is increased from 3.2 to 5.2. The presence of metal cations such as Na⁺, K ⁺, Ca²⁺, Mg²⁺ and other transition metal cations does not interfere with its fluorescent pH response. In addition, the intracellular pH fluorescent imaging ability of the probe has been confirmed on macrophage cells using a confocal microscope. The Royal Society of Chemistry and the Centre National de la Recherche Scientifique.

Full text of DOI:10.1039/b9nj00703b

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PAPER
www.rsc.org/njc | New Journal of Chemistry
A charge transfer type pH responsive fluorescent probe and its
intracellular applicationw
Zhipeng Liu,ab Changli Zhang,ad Weijiang He,*a Fang Qian,a Xiaoliang Yang,a
Xiang Gaoc and Zijian Guo*ab
Received (in Montpellier, France) 24th November 2009, Accepted 25th November 2009
First published as an Advance Article on the web 4th February 2010
DOI: 10.1039/b9nj00703b
A new charge transfer pH fluorescent probe BTP has been prepared by the ethylene bridging of
benzothiazole and pyridine. The probe exhibits a specific fluorescent response to pH with a large
Stokes shift, and the pH-induced emission enhancement factor (EEF) is about 22-fold when the
pH is increased from 3.2 to 5.2. The presence of metal cations such as Na+, K+, Ca2+, Mg2+
and other transition metal cations does not interfere with its fluorescent pH response. In addition,
the intracellular pH fluorescent imaging ability of the probe has been confirmed on macrophage
cells using a confocal microscope.
construct intramolecular charge transfer (ICT) fluorescent
probes displaying a large Stokes shift.18 Rurack and coworkers
Introduction
À
Intracellular pH plays a central role in many cellular events,
such as cell growth, endocytosis, cell adhesion and other
cellular processes.1–4 Determination of pH by fluorescent
methods is attracting much more attention in pH imaging
and sensing due to the advantages over other techniques
including rapid response time, high signal-to-noise ratio, non-
invasiveness, and excellent pH sensitivity.5,6 In general, there
are two types of synthetic pH probes, one type for cytosol that
work at a pH of 6.8–7.4,5,7–12 and another type for the acidic
organelles (for example, lysosomes) functioning in the pH
range 4.5 to 6.0.13–16 Although lots of the former type of
probe have been reported, little attention has been paid to the
latter. In fact, the abnormal pH values in acidic organelles
have been reported to be closely associated with some cellular
dysfunction. The defective pH regulation of acidic compart-
ments in human breast cancer cells and the elevated lysosome
pH in neural ceroid lipofuscinoses are the well known
examples.17 On the other hand, many fluorescent pH probes
still suffer from the severe excitation interference caused by a
shorter Stokes shift.14,15 Therefore, the development of specific
pH fluorescent probes of large Stokes shift for the acidic
pH range is appealing to the fields of chemistry, cytology
and pathology.
have reported an ICT sensor for H2PO4 via ethylene
bridging of the bisamidopyridine receptor and benzothiazole.
Moreover, ICT sensors for cations have also been constructed
by bridging azocrown ether and benzothiazole. All these
sensors display a large Stokes shift (more than 100 nm).18a,b
In this work, a new fluorescent pH probe for acidic micro-
environments, BTP, was constructed by ethylene bridging of
benzothiazole and pyridine to form a D–p–A fluorophore.
Herein, pyridine was introduced as the H+ binding moiety
since the low pKa value (5.3 at 20 1C) of the pyridine N favors
H+ binding in acidic conditions.19 In addition, its benzo-
thiazole motif, which has a low affinity for H+ and metal
cations, is of benefit to the specific pH response of probe.8 The
ethylene bridge between benzothiazole and pyridine makes
BTP an D–p–A type fluorophore with the dimethylamino
group and benzothiazole/pyridine acting respectively as D
and A, favoring a large Stokes shift and reducing the excitation
interference (Scheme 1).
Benzothiazole derivatives with a D (donor)–p–A (accepter)
structure type are frequently adopted as the fluorophore to
a State Key Laboratory of Coordination Chemistry, Coordination
Chemistry Institute, School of Chemistry and Chemical Engineering,
Nanjing University, Nanjing 210093, P. R. China.
E-mail: heweij69@nju.edu.cn, zguo@nju.edu.cn;
Fax: +86 25 83314502; Tel: +86 25 83597066,+86 2583686218
b State Key Laboratory of Bioelectronics, Southeast University,
Nanjing 210096, P. R. China
c Animal Model Research Center, Nanjing University, Nanjing 210093,
P. R. China
d Nanjing Xiaozhuang College, Nanjing 210017, P. R. China
w Electronic supplementary information (ESI) available: Character-
ization of BTP and photospectroscopic study. See DOI: 10.1039/
b9nj00703b
Scheme 1 Synthesis of BTP.
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Results and discussion
Synthesis of BTP
BTP was prepared simply in a good yield (78%) by stirring the
mixture of picolinaldehyde and 6-(N,N-dimethyl)amino-2-
methylbenzothiazole (1) in the presence of KOH at room
temperature for 24 h. Compound 1 was synthesized from
2-methylbenzothiazole following a reported procedure under-
going nitration, reduction and methylation in sequence.20
Spectroscopic properties and optical responses to pH
The fluorescence and UV–vis pH titration of BTP were
performed in aqueous media (containing 1% methanol)
over a pH range from 3 to 12. Free BTP in aqueous solution
(pH = 6.02) exhibits an intense emission band centered at
596 nm (lex = 397 nm) with a large Stokes shift of 199 nm in
neutral conditions (Fig. S7 of the ESIw). Its quantum yield in
neutral conditions was determined as 0.22 with quinine sulfate
in 0.5 M H2SO4 as per ref. 21. The large Stokes shift should help
to reduce the excitation interference. In fact, BTP displays
stable emission when the pH value is higher than 5.2 (Fig. S8
and S9 of the ESIw). However, BTP shows very weak fluores-
cence when the pH value is lower than 3.1. Distinct emission
enhancement can be observed without emission shift when the
pH is raised from 3.2 to 5.2, and the emission enhancement
factor is around 22 [Fig. 1(a)]. Fig. 1(b) illustrates the fluore-
scent pH titration profile according to the emission intensity at
597 nm. Fitting the profile with the Henderson–Hasselbach
equation gives a pKa of 4.22 for BTP.22 This pKa value almost
matches the typical pH values of acidic organelles such as
lysosomes, suggesting BTP might be suitable for detecting the
altered pH of lysosomes.
Fig. 1 (a) Emission spectra of BTP (5 Â 10À6 M) in aqueous
solutions (MeOH–water = 1 : 99, v/v) at different pH, (b) emission
intensity at 597 nm versus pH in acidic conditions according to the
fluorescent pH titration. lex = 397 nm.
The addition of transition metal cations such as Hg2+
,
Cd2+, Pd2+, Cu2+, Ni2+, Mn2+, Zn2+, Fe2+ and Ag+ to
BTP solution does not lead to any notable emission change
(Fig. 2). The results confirm the specific fluorescent response of
BTP to H+. In addition, the presence of Na+, K+, Ca2+ and
Mg2+, which are abundant in cells, at a concentration of 1000
times as that of other metal cations does not interfere with the
pH sensing behaviour of BTP in acidic environments. All these
results suggest that BTP might be a suitable candidate as a
staining agent for fluorescent intracelluar pH imaging.
1H NMR spectra of BTP at different pH values have been
determined in CD3OD/D2O (10 : 1, v/v) (Figs. 3 and 4). The
results disclose that the chemical shifts of BTP protons remain
almost constant when the pH is higher than 4.35 (pD values
have been converted into pH values).23 When decreasing the
pH from 4.35 to 3.72, one can see the clear down-field shift for
the signals of the pyridine protons Hd, Hb and Hc. The Hd
signal shifts from 7.36 to 7.38, Hb from 7.67 to 7.70 and Hc
from 7.86 to 7.89. These results suggest that the protonation of
pyridine N (N2) may occur in this process, although the down-
field shift of Ha is very minor. The constant chemical shift of
the dimethylamino group protons (Hi) in this process implies
that the amino N (N1) is still not protonated even at pH 3.72.
The distinct down-shift of all BTP protons can only be
observed when the pH becomes lower than 2.99. The 1H
NMR titration implies that the emisson enhancement of
Fig. 2 Emission change at 597 nm of BTP (5 Â 10À6 M) in aqueous
solution induced by different metal cations (1 : 99, MeOH–water, v/v;
50 mM HEPES, 100 mM KNO3; pH 7.40), lex = 397 nm. The final
concentration of Hg2+, Cd2+, Pb2+, Cu2+, Ni2+, Mn2+, Zn2+
Fe2+, Hg2+ and Ag2+ is 5 mM, respectively, while that of Na+
K+, Ca2+ and Mg2+ is 5 mM, respectively.
,
,
BTP from pH 3.2 to 5.2 should be ascribed to the deproto-
nation of pyridininum. In fact, the pyridine N protonation-
induced emission quenching has also been reported by other
scientists.24 The protonation of the dimethylamino group at
pH o3.0 may destroy the ICT effect of the BTP molecule,
which makes BTP become non-fluorescent. The N atom (N3)
in the benzothiazole ring is difficult to protonate due to its lone
electron pair being involved in the aromatic conjugation
system.25,26
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Fig. 5 Absorption spectra of BTP (5 Â 10À6 M) in aqueous solutions
(MeOH–water, 1 : 99, v/v) at different pH values. The arrows display
the variation of absorbance from pH 3.86 to 5.83.
Fig. 3 1H NMR spectra of BTP in CD3OD/D2O (10 : 1, v/v) at
different pH values.
microscopy. The macrophage cells after 10 min of incubation
with BTP (10 mM, in PBS) for 10 min at 37 1C show
punctuated fluorescence inside the cells especially near
the nucleus and cell membrane, displaying the preferential
affinity of BTP for lysosomes upon excitation at 405 nm
(Fig. 6b and Fig. S11). The subsequent single rinse with PBS
(pH 7.4) induces only a very minor decrease of fluorescence in
the macrophage cells (Fig. 6c), suggesting BTP is difficult to
exclude from macrophage cells by rinsing. However, the
fluorescence in macrophage cells can be distinctly depressed
(Fig. 6d) by incubating the cells with ATP (1 mM) for 3 min.
The distinct emission decrease is consistent with the fact that
the extracellular ATP stimulation normally induces the pH
decrease of lysosomes.27 The fine cell permeability and
effective imaging ability for the pH variation around pH 4
indicate that BTP is an effective intracellular pH
imaging agent.
Fig. 4 pH Titration profiles of BTP according to the chemical shifts
of the pyridine protons (a) and N,N-dimethylamine protons (b) at
different pH values (pH values were converted from pD by pH =
pD–0.4).23
The UV–vis titration of BTP from pH 3.86 to 5.0 exhibits
an increase in the p–p* transition band at 390 nm which is
accompanied by a decrease in the broad ICT band (at 417 nm)
and in a minor band at 341 nm (Fig. 5). The clear isobestic
points at 441 nm and 350 nm imply that only one reaction
occurred from pH 3.86 to 5.0, which can be ascribed to the
conversion of free BTP to the protonated [BTP+H+] accord-
ing to the 1H NMR titration data. On the other hand, the UV
spectra of BTP exhibit a stable maximal absorption band
centered at 390 nm (e = 2.23 Â 104 MÀ1 cmÀ1) at pH 5–12
(Fig. S10 of the ESIw) similar to the stable emission band
above pH 5.
Fig. 6 Confocal fluorescence imaging of macrophage cells. (a) Bright-
field transmission image of the cells stained with BTP (10 mM, PBS
solution) at 37 1C for 10 min; (b) fluorescence image of (a);
(c) fluorescence image of cells in (b) after rinsing once with PBS
solution for 3 min (200 mL); (d) fluorescence image of macrophage
cells in (c) followed by further treatment with 1 mM ATP solution for
3 min. lex, = 405 nm; band path for detection = 550–650 nm.
Intracellular pH imaging with BTP as the imaging agent
The intracellular pH imaging ability of BTP has been investi-
gated on macrophage cells by laser scanning confocal
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1H NMR titration
Conclusions
1
The H NMR study of the pH titration were carried out on
Bruker DRX-500 (500 MHz) by adding DCl or NaOD in
D2O to the solutions of BTP (8 Â 10À3 M) in CD3OD: D2O
(10 : 1, v/v) to modulate the different pH values. Chemical shift
was referenced to an external sample of TMS (d = 0.00 ppm).
The experiments were carried out at 298 K.
In summary, a D–p–A type fluorescent pH probe BTP has
been synthesized by linking the 2-pyridine motif to benzothia-
zole. This probe has a pKa of 4.22 and a remarkable emission
enhancement is observed when increasing the pH from 3.2 to
5.2. The large Stokes shift, fine cell permeability and specific
pH fluorescent response make BTP an effective intracellular
pH imaging agent for acidic microenvironments. The current
study demonstrates that the conjugation of the fluorophore
with pyridine to form an ICT fluorophore might be an
effective strategy to construct new fluorescent pH probes of
large Stokes shift for acidic microenvironments.
Selective fluorescence response of BTP to different metal cations
Stock solutions of the metal ions (1.2 mM) were prepared in
deionized water. The fluorescence response of BTP to different
metal cations was determined in 5 mM BTP buffered solution
(1 : 99, MeOH–water, v/v; 50 mM HEPES, 100 mM KNO3;
pH 7.40). Metal cation solution (12.5 mL, 1.2 mM) was added
to 3 mL of this solution, and the fluorescence spectra were
determined after complete mixing. The excitation wavelength
was 397 nm. The final concentration of alkaline or alkaline
earth metal cation was 1000 times as high as that of other
cations.
Experimental sections
Materials and general methods
All reagents were of analytic grade. The stock solutions of
metal ions for fluorescence discrimination were prepared
respectively by dissolving MnCl2, PbCl2, FeCl2, Zn(NO3)2Á
7H2O, CaCl2, NaCl, CuSO4, NiCl2Á6H2O, KCl, CdCl2Á
2.5H2O, HgCl2, MgCl2Á6H2O, AgNO3 in doubly distilled
water. The 1H NMR and 13C NMR spectra were recorded
on a Bruker DRX-300 spectrometer with TMS as the internal
standard in CDCl3 or CD3OD/D2O. Mass spectrometric data
were determined with a LCQ (ESI-MS, Thermo Finnigan)
mass spectrometer. Elemental analyses for C, H and N were
performed on a Perkin-Elmer 240 C analyzer. Fluorescence
measurements were performed on an AMINCO Bowman
series 2 with 3 nm slit for both excitation and emission. The
fluorescence quantum yield was determined using quinine
sulfate solution (F = 0.546 in 0.5 M H2SO4) as per ref. 21.
Absorption spectra were measured on a Shimadzu UV-3100 or
an UV–vis–NIR spectrophotometer. All pH and pD measure-
ments were determined using a Model PHS-3C meter.
Cell culture and intracellualr pH fluorescence imaging
Murine peritoneal macrophages were isolated from 6- to
12-week-old C57BL/6J mice by using a reported protocol.28
After culturing on coverslips for 2 h, the floating cells were
removed by extensive washing, and the attached cells were
maintained in RPMI 1640 medium (Invitrogen) containing
10% fetal bovine serum, penicillin (100 units/mL), strepto-
mycin (100 mg mLÀ1) and 5% CO2 at 37 1C. After removing
the incubation media and rinsing with PBS three times, the
cells were stained by incubating the cells in 10 mM BTP
solution for 10 min at room temperature. Then the cells were
imaged with an Olympus FV1000 microscope equipped with a
60 Â 1.40 NA oil-immersion objective. After removing the
BTP media, the cells were treated with PBS for 3 min followed
by imaging. Then the PBS solution was removed and the cells
were cultured with BTP for 10 min followed by rinsing with
1 mM ATP solution for 3 min. Then the cells were imaged
again. For all imaging, the samples were excited at 405 nm,
and the band path was 550–650 nm.
Sample preparation for UV–vis and fluorescence determination
A stock solution of BTP (0.5 mM) was prepared in MeOH.
The BTP solution for spectroscopic determination was
obtained by diluting the stock solution to 5 mM with deionized
water. In the titration experiments, 3 mL of BTP solution
(5 mM, 0.5 mM) was poured into a quartz optical cell of 1 cm
optical path length each time and the slight pH variations of
the solution were achieved by adding the minimum volumes of
NaOH or HCl. Spectral data were recorded immediately after
each addition. To determine the fluorescence response of BTP
to different metal cations, the stock solution of BTP
was diluted to 5 mM with HEPES solution (50 mM HEPES,
100 mM KNO3; pH 7.40).
Synthesis
A mixture of 6-(N,N-dimethyl)amino-2-methylbenzothiazole
(0.70 g, 3.65 mmol), picolinaldehyde (0.50 g, 3.65 mmol) and
powdered KOH (0.93 g, 16.6 mmol) in 3 mL DMF was stirred
at 25 1C for 24 h. Then, the mixture was partitioned between
H2O (5 mL) and CH2Cl2 (10 mL). The aqueous portion was
extracted with CH2Cl2 (3 Â 10 mL). Then, the combined
organic extracts were dried over anhydride MgSO4 followed
by removing the solvent via evaporation in vacuo. The crude
product was purified on silica gel (EtOAc: CH2Cl2 = 1 : 10)
to afford BTP as red powder. Yield 0.80 g (78%); m.p.
148–149 1C. Elemental analysis, found: C, 68.23; H, 5.48; N,
15.12; calc. for C16H15N3S: C, 68.30; H, 5.37; N, 14.93. 1H
NMR (CDCl3, 300 MHz), d (ppm): 3.06 (s, 6H, BT–N(CH3)2),
6.94–6.98 (dd, 1 H, BT–H), 7.09 (d, 1H, J = 5.1 Hz BT–H),
7.20–7.24 (m, 1H, Py-H), 7.48–7.51 (d, 1H, J = 7.8 Hz,
BT–H), 7.69–7.75 (m, 1 H, Py–H), 7.44–7.49 and 7.79–7.85
UV–vis and fluorescence pH titration
The UV titration experiments on BTP were carried out by
adding the diluted HCl or NaOH aqueous solution to 3 mL of
BTP solution (5 mM, 1 : 99, MeOH–water v/v) in a cuvette.
The spectra and pH values were recorded after the solution
was completely mixed. The fluorescence titration experiment
was investigated in a similar procedure and the excitation
wavelength was 397 nm.
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(2 Â d, 2H, J = 15.2 Hz, –CHQCH–), 7.88 (d, 2 H, J = 9.3 Hz,
Py–H), 8.66 (d, 1H, J = 4.8 Hz, Py–H). 13C NMR (CDCl3,
75 MHz), d (ppm): 39.62, 102.28, 113.52, 122.40, 122.96,
123.31, 125.67, 133.95, 136.62, 137.34, 149.26, 149.69,
153.87, 161.49. ESI-MS (positive mode, m/z): Calcd. 282.11,
found: 282.19 for [M+H]+.
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Acknowledgements
We thank the National Science Foundation of China (Nos.
20871066, 20631020, 90713001, 10979091 and 20721002) and
the Natural Science Foundation of Jiangsu (BK2008015,
BK2009227) for financial support. This work is also supported
by the Open Fund of State Key Laboratory of Bioelectronics,
Southeast University.
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