A novel coumarin derivative as a sensitive probe for tracing intracellular pH changes

Article abstract of DOI:10.1039/c4ra11347k

A new pH sensitive probe, 7-diethylamino-3-[2-(4-piperidin-1-yl-phenyl)-vinyl]-chromen-2-one (CS-P), was designed and synthesized. This probe was constructed by introduction of piperidine to the coumarin stem. It exhibited high pH sensitivity ranging from pH 5.9 to pH 3.0 (pK_a = 4.55), and in vitro cell imaging showed that its fluorescence intensity was distinctly related to the cellular pH changes induced by chloroquine or dexamethasone. Demonstrated as having good cell membrane permeability and high pH sensitivity, this probe is suitable for tracing the intracellular pH changes from neutral to acidic conditions. This journal is

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A novel coumarin derivative as sensitive probe
for tracing intracellular pH changes
Cite this: DOI: 10.1039/x0xx00000x
Mengqiang Liua*, Minshan Huc*, Qian Jianga, Zhiyun Lua, Yan Huanga, Yanfei
Tan*b, Qing Jiang*a,b
Received 00th January 2012,
Accepted 00th January 2012
A new pH sensitive probe, 7-diethylamino-3-[2-(4-piperidin-1-yl-phenyl)-vinyl]-chromen-2-one
DOI: 10.1039/x0xx00000x
(
CS-P), was designed and synthesized. This probe was constructed by introduction of piperidine
to the coumarin stem. It exhibited high pH sensitive ranged from pH 5.9 to pH 3.0 (pKa = 4.55), in
vitro cell imaging showed that its fluorescence intensity was distinctly related to the cellular pH
changes induced by chloroquine or dexamethasone. Demonstrated with good cell membrane
permeability and high pH sensitivity, this probe is suitable for tracing the intracellular pH changes
from neutral to acidic conditions.
www.rsc.org/
Introduction
Fluorescent probe offers a unique approach for visualizing
morphological details of tissues with subcellular resolution and
Intracellular pH plays key roles in modulating cell growth and
differentiation, enzyme and tissue functions, such as cell cycle
control,1 cellular proliferation and apoptosis,2 membrane
potential and ion regulation,3 muscle contraction4 and multidrug
resistance (MDR).5
has become a powerful tool for manipulating and investigating
living cells and even animals.11-14 There are different kinds of
fluorescent probes with various skeleton structure, including
rhodamine,15 BODIPY,16 fluorescein,17 tricarbocyanine,18
benzo[a]phenoxazine,19
coumarin20
etc.
From
these
Abnormal pH values are associated with inappropriate cell
functions, growth, and division, which can cause many serious
human diseases. An enhanced activity of Na(+)/Li(+)
fluorophores, some special probes with pH sensitivity can be
designed and synthesized by changing functional groups to
these skeletons.
countertransport, studied as
a surrogate of Na(+)/H(+)
Coumarins are a group of good candidates, because they
have very high fluorescence quantum yield,21 large stokes
shift,22 excellent light stability, and low toxicity.23 At the same
time, coumarin derivatives have expanded emission profiles
tunable from the blue to near-infrared (NIR) region by simply
changing donors at either the 6- or 7-position and the electron
acceptor at either 3- or 4- position of the coumarin core
structure.24 So far, based on the coumarin platform, a number of
pH sensitive fluorescent probes with different functional groups
had been constructed.25-27 However, connecting H+ binding
sites to the coumarin skeleton by styrene bond for increasing
conjugated system it has not been reported.
exchanger, has been described in red blood cells of patients
with cardiac syndrome X.6 Mucolipidosis type IV (MLIV) is a
neurodegenerative channelopathy and in MLIV the lysosomal
pH is lower than normal.7 Eukaryotic cell contains some
compartments with different acidity degrees, for example, the
cytoplasma is slightly alkaline at about pH 7.2, whereas some
organelles, such as lysosomes and endosomes, have
intracompartmental pHs of 4.0-6.0.8 Lysosomes and endosomes
which participate in the critical functions of endocytic and
digestive processes have pH value of 4.7 and 5.5, respectively.9
Therefore, monitoring of pH changes in live cells is critical for
studying cellular functions and further understanding
physiological and pathological processes.
In this paper we developed a new coumarin derivative,
termed 7-diethylamino-3-[2-(4-piperidin-1-yl-phenyl)-vinyl]-
chromen-2-one (CS-P), and its photophysical properties and
intracellular pH sensitivity were studied.
There are many popular techniques to measure intracellular
pH, including microelectrodes, nuclear magnetic resonance
(NMR), and molecular spectroscopy. Among these techniques,
fluorescence spectroscopy has attracted much more attention
due to its excellent sensitivity, fast response time, high signal-
to-noise ratio and the ability to continuously monitor the rapid
kinetic pH changes.10
Experimental
Materials and methods
All chemicals and solvents were at analytical grade and freshly
distilled prior to use without further purification, except
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spectrographically pure dimethylsulfoxide (DMSO) used for
2
(0.3 g, 1.01 mmol), appropriate styrene (1.1 eq), tris(o-
biological evaluation. Chloroquine was commercially available tolyl) phosphine (12.28 mg, 0.0404 mmol), palladium acetate
from Aldrich (St.Louis, MO) and used without further (4.52 mg, 0.0202 mmol) and Et3N (1.97 mL, 14.14 mmol) were
purification. Double distilled water was used for preparation of added in 24 mL of argon degassed DMF in a flask. After
the buffer solutions. 1H NMR and 13C NMR spectra were stirring at 100 ºC for 24 h under argon, the mixture was cooled
measured by BrukerAvance AV ΙΙ-400 MHz spectrometer. FT- to room temperature and poured into water. Resulting solid was
IR spectra were recorded on a Perkin-Elmer 2000 infrared filtered off and washed with water, then dried. The crude
spectrometer with KBr pellets under an ambient atmosphere. product residue was purified by column chromatograph over
UV-Vis absorption spectra were performed on a Perkin Elmer silica to yield the pure product.
Lambda 950 UV/VIS Spectrometer. Fluorescence spectra were
7-diethylamino-3-styryl-chromen-2-one（CS）
collected
on
a
PerkinElmer
LS55
fluorescence
spectrophotomete using excitation wavelength of 400 nm. The Orange red solid. Recrystallized from ethanol. Yield: 46.9 %;
pH values were determined with PHS-3E pH Meter calibrated 1H NMR (400 MHz, CDCl3) δ (ppm): 7.68 (s, 1 H, ArH), 7.52
at room temperature (25
±
2ºC) with standard buffers of pH (d,
J
= 7.6 Hz, 2 H, ArH), 7.47 (d,
= 7.2 Hz, 2 H, ArH), 7.30 (d,
= 7.2 Hz, 1 H, ArH), 7.11 (d,
J
= 16.0 Hz, 1 H, =CH), 7.36
6.86 and 4.00. Acetic acid and sodium acetate were used for (t,
tuning pH values. Fluorescence microscopic images were (d,
J
J
= 8.8 Hz, 1 H, ArH), 7.24
J
J
= 16.0 Hz, 1 H, =CH), 6.60
obtained from High-speed Widefield Live-cell System (Leica (dd,
J
= 8.8 Hz,
J
= 2.4 Hz, 1 H, ArH), 6.51 (d,
J = 2.4 Hz, 1 H,
AF7000).
ArH), 3.45-3.39 (m, 4 H, -CH2), 1.23 (t,
J
= 6.8 Hz, 6 H, -CH3).
13C NMR (100 MHz, CDCl3): δ (ppm) 161.5, 155.6, 150.4,
138.0, 137.6, 130.0, 128.8, 128.7, 127.6, 126.6, 123.1, 117.8,
Synthesis
The detailed synthetic routes were shown in Scheme 1. 4- 109.1, 109.0, 97.2, 44.9, 12.5. IR (KBr), cm-1: 3070, 3021,
piperidin-1-yl-benzaldehyde28 and 7-diethylamino-chromen-2- 2975, 2920, 1704, 1616, 1587, 1523, 1238, 1139, 975, 750. MS
one29 were synthesized according to the literature procedures.
(ESI)+: m/z 320.1644 (M + H) +; calcd for (M + H)+: 320.1651.
Synthesis of 1-(4-vinyl-phenyl)-piperidine (1)
7-diethylamino-3-[2-(4-piperidin-1-yl-phenyl)-vinyl]-chromen-2-
one (CS-P)
n-BuLi (3.2 mL of 2.5 M in n-hexane) was added dropwise to a
1
stirred solution of Ph3PCH3I (3.2 g, 7.85 mmol) in THF (25 Yellow solid. Yield: 56.1 %; H NMR (400 MHz, CDCl3) δ
mL) at 0 °C under inert atmosphere. After stirring for 30 min, a (ppm): 7.63 (s, 1 H, ArH), 7.42 (d,
J
= 8.4 Hz, 2 H, ArH), 7.36
= 8.0 Hz, 1 H, ArH), 6.97
= 8.4 Hz, 2 H, ArH), 6.59
solution of 4-piperidin-1-yl-benzaldehyde (5.23 mmol) in THF (d,
(10 mL) was added dropwise and the resulting mixture was (d,
J
J
= 16.0 Hz, 1 H, =CH), 7.28 (d,
= 16.0 Hz, 1 H, =CH), 6.90 (d,
J
J
stirred at room temperature for 10 h. Then, saturated (dd,
ammonium chloride was added and followed by extracting with ArH), 3.44-3.38 (m, 4 H, -CH2), 3.22 (t,
PE. The organic layer was washed with brine before being 1.73-1.67 (m, 4 H, -CH2), 1.62-1.57 (m, 2 H, -CH2), 1.23 (t,
J
= 8.8 Hz,
J
= 2.0 Hz, 1 H, ArH), 6.51 (d,
J = 1.6 Hz, 1 H,
J
= 5.2 Hz, 4 H, -CH2),
J
=
dried over anhydrous magnesium sulfate. After evaporating off 7.2 Hz, 6 H, -CH3). 13C NMR (100 MHz, CDCl3): δ (ppm)
the solvent, the residue was purified by column 161.7, 155.3, 151.5, 150.0, 136.3, 129.9, 128.5, 128.2, 127.6,
chromatography on silica gel to give a pure yellow solid (Yield: 119.6, 118.6, 115.9, 109.3, 109.0, 97.2, 50.1, 44.8, 29.7, 25.7,
1
85.0 %). H NMR (400 MHz, CDCl3) δ (ppm): 7.31 (d,
J
= 8.4 24.4, 12.5. IR (KBr), cm-1: 2971, 2925, 2849, 1704, 1594,
= 8.4 Hz, 2 H, ArH), 6.67-6.60 (m, 1 1511, 1232, 1128, 810. MS (ESI)+: m/z 403.2383 (M + H) +;
= 7.2 Hz, 1 H, =CH2), 5.07 (d,
= 10.8 Hz, calcd for (M + H)+: 403.2386.
= 5.4 Hz, 4 H, -CH2), 1.73-1.68 (m, 4 H,
-CH2), 1.61-1.58 (m, 2 H, -CH2).
Hz, 2 H, ArH), 6.89 (d,
H, =CH), 5.58 (d,
1 H, =CH2), 3.18 (t,
J
J
J
J
Solvatochromic effects of CS and CS-P
CS CS-P (8 ꢀM) were prepared in cyclohexane, toluene,
tetrahydrofuran, acetonitrile or dimethyl sulfoxide, respectively.
,
Synthesis of 3-bromo-7-diethylamino-chromen-2-one (2)
Br2 (1.4 g, 9.2mmol) was added dropwise to a solution of 7- Absorption spectra and fluorescence emission spectra of CS
diethylamino-chromen-2-one (2.0 g, 9.2 mmol) in acetic acid CS-P in different solvents were recorded (λex = 400 nm).
(40 mL), and the mixture was stirred at room temperature for 2
,
Fluorescence changes of CS-P under different pH values
h. After completion of the reaction, the precipitate was filtered
off and washed with acetic acid, then dried under vacuum and CS and CS-P (4 ꢀM) were prepared in DMSO-water (1:1, v/v)
recrystallized in acetonitrile to afford a light yellow solid buffer solution. These solutions were modulated by acetic acid
1
(Yield: 80.6%). H NMR (400 MHz, DMSO-d6) δ (ppm): 8.34 and sodium acetate to achieve different pH values (3.0 ~ 7.6)
(s, 1 H, ArH), 7.44 (d,
Hz, = 2.4 Hz, 1 H, ArH), 6.56 (s, 1 H, ArH), 3.45-3.41 (m, 4 solutions were kept at ambient temperature for 1-2 h, the
H, -CH2), 1.13 (t,
J = 8.8 Hz, 1 H, ArH), 6.75 (dd, J
= 8.8 with maintaining Na+ concentrtion of 0.1 mol/L. After the
J
J
= 7.2 Hz, 6 H, -CH3).
fluorescence emission spectra were recorded (λex = 400 nm).
The constant pKa of CS-P was calculated based on the
following formula, Fmin and Fmax represented the fluorescence
General procedure for the synthesis of target compound
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intensity at minimal and maximal H+, respectively, and
the stoichiometry of H+ binding to the probe.
n meant
Fmax[H+]n+FminKa
F
Ka+[H+]n
In addition, absorption spectra of CS-P (4 ꢀM) in a DMSO-
water solution (1:1, v/v) with different pH values were
recorded.
The selectivity of CS-P for H+
Considering that nitrogen and oxygen could bind many metal
ions in solution, it was very important to determine whether
other ions were potential interferents to CS-P. The metal ions
and biologically relevant analytes, Zn(NO3)2, MgSO4, NaCl,
MnSO4, BaCl2, KCl, CaCl2, CuSO4, FeCl3, Cr(NO3)3, FeSO4,
NiSO4, HgCl2, Pb(NO3)2, L-cystine and reduced glutathione,
with the same concentration of 40 ꢀM, were added as
interferents to the solution (DMSO:H2O = 1:1, v/v) of CS-P
(ultimate concentration: 4 ꢀM). Fluorescence emission spectra
of these solutions were recorded with excitation at 400 nm.
Scheme 1 The synthetic routes of the objective compounds. (i) BuLi, Ph3PCH3I,
THF, 0 °C; (ii) Br2, CH3COOH; (iii) Pd(OAC)2, P(O-toly)3, Et3N, DMF, 100 °C.
Solvatochromic Effects
Solvatochromism of CS
,
CS-P were investigated in several
CS-P were
solvents. The photophysical properties of CS
,
compiled in Table S1. The absorption spectra and the
fluorescence emission spectra of CS and CS-P in various
solvents were shown in Figure S1 and Figure S2, respectively.
The λabs(max) and λem(max) values of CS-P were strongly
affected by solvent polarity. These results showed that
piperidine, the functional group on the CS-P, aroused of a
bathochromic shift of 25-40 nm, compared with CS. A large
stokes shift (>75 nm) and solvent-dependent emission spectra
indicated that effective intramolecular charge transfer (ICT) had
occurred in the excited state between the coumarin unit and the
substituent at position 3.
Tracing intracellular pH changes
Mouse macrophages Raw 264.7 cells were cultured in 24-well
plate in this paper and were internalized with 2 ꢀM CS-P for 15
min at 37 ºC, then washed three times with PBS buffer to
remove excess probe, chloroquine (100 ꢀM) was added to
stimulate lysosomal pH change based on the reference. 15 Cells
were observed under High-speed Widefield Live-cell System
(Leica AF7000) from 0 minute to 60 minute with a time
interval of 3 minutes.
Spectral studies of CS-P
HeLa cells were co-cultured with CS-P (2 ꢀM) in 24-well
plate for 15 min, then washed with fresh DMEM medium and
dexamethasone (2 ꢀM) was added to decreasing the cellular
pH. Cells were observed and recorded by High-speed Widefield
Live-cell System (Leica AF7000) from 0 minute to 60 minute
with a time interval of 3 minutes.
The absorption spectra, excitation spectra and the fluorescence
spectra of CS-P at different pH values were shown in Figure
S3(A), Figure S3(C) and Figure 1(A), respectively. Along with
decreasing of the pH value from 7.6~3.0, the absorbance was
significantly enhanced at 443 nm. In the meanwhile, the color
of the solution changed from dark yellow to bright yellow-
green, which indicated that CS-P could serve as a visual
indicator for H+ concentration (Figure S3(B)). Figure 1(A)
showed that only very weak fluorescence intensity was
observed in pH = 5.9~7.6 solution. However, the fluorescence
intensity was significantly enhanced with pH values decreased
from pH 5.9 to 3.0. The intensity of CS-P in pH 3.0 showed
more than 47 fold higher compared with in pH 5.9, while the
fluorescent intensity of the control compound, CS, did not
changed in different pH values (Figure S4.). The analysis of
emission intensities changed at 515 nm as a function of pH
obtained a pKa of 4.55 (Figure 1(B)), the pKa value almost
matched with the typical pH value of the acidic organelles
(such as lysosomes and endosomes), which indicated that CS-P
could be suitable for monitoring pH variations in the acidic
Furthermore, in order to determine the cellular location of
CS-P, MG63 cells were co-stained with LysoTracker® Red
(0.1 ꢀM) (L7528, Invitrogen) or MitoTracker® Deep Red (0.1
ꢀM) (M22426, Invitrogen), respectively.
Results and discussion
Synthesis
The new coumarin derivative, CS-P, was synthesized from 1-
(4-vinyl-phenyl)-piperidine
and
3-bromo-7-diethylamino-
chromen-2-one shown in Scheme 1. Their structures were
confirmed by 1H NMR, 13C NMR. 7-diethylamino-3-styryl-
chromen-2-one (CS) was synthesized and used as control to
reveal the primary mechanism of fluorescence changes of CS-P
in the different pH condition, the structure difference between
CS and CS-P was that CS-P was constructed by piperidine
group on the stem of coumarin. Piperidine derivatives were
effective proton receptors with different pKa values depending
on the skeletons and position of substituents on the piperdine
group. 30, 31
environments (pH 4-6) in vitro
.
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experimental condition unchanged at pH 7.2, the results (Figure
3) showed these cations did not increase the fluorescent
intensity of CS-P. Moreover, amino acids (Cys and GSH) also
exhibited negligible effects on the activity of CS-P, only a
certain amount of H+ could lead to remarkable fluorescence
enhancement. The competition experiments of CS-P revealed
that the H+-depended fluorescence response was unaffected in
the presence of background ions (Figure S5). The fluorescent
intensity of CS-P could be reversibly selected by
protonation/deprotonation of the piperidine moiety under acidic
or alkaline condition (Figure S6). In addition, it exhibited high
photostability (Figure S7). The high selectivity of CS-P over
interferents, along with its photostability and reversibility,
indicated CS-P had considerable potential as practical pH
probe.
Figure 1 (A) Fluorescence spectra of CS-P (4 μM) in various pH (7.6-3.0) in DMSO-
water (1:1, v/v) solution (λex = 400 nm). (B) Sigmoidal fitting of the pH-dependent
fluorescence intensity at 515 nm. Inset: the good linearity in the pH range of 3.2-
5.9.
The switching mechanism
CS-P and its protonated form of HCl were prepared to study
the proton-binding behavior by 1H NMR spectroscopy. As
shown in Figure 2(A), Hb and Hc displayed a slight downfield
shift of 0.05 and 0.04 ppm, respectively, suggesting that the
diethylamine on the CS-P did not bind H+. However Ha
displayed significant downfield shifts of 0.74 ppm, indicating
that the protonation occurred at the piperidine site. The possible
mechanism of pH sensitivity of CS-P was illustrated in Figure
2(B). Under neutral pH condition (pH = 7.2), CS-P existed
intramolecular charge transfer (ICT) process from piperidine
part to coumarin matrix and weak fluorescence was due to
strongly affected by solvent polarity. In contrast, under acidic
pH condition (pH = 4.3), the nitrogen atom of piperidine group
was protonated, which inhibited the ICT process and led to
fluorescence enhancement.
Figure 3 Fluorescence response (515 nm) of CS-P interfered by different ions and
amino acids in DMSO-water (1:1, v/v) solution.
Tracing intracellular pH changes
The cytotoxicity of CS-P was evaluated by the MTT assays.
CS-P showed no cytotoxicity (Figure S8).
In vitro cell imaging was carried by employing CS-P to
monitor intracellular pH changes induced by chloroquine in
Raw 264.7 cells. Chloroquine could increase intracellular pH in
macrophage cells.32 CS-P exhibited strong yellow fluorescence
in these cells before treated with chloroquine, the fluorescence
intensities reduced distinctly in the cells by addition of
chloroquine according to continuous observation within 60 min
(Figure 4).
Dexamethasome could induce cell apoptosis and
intracellular acidification was happened spontaneously.15 After
treatment with dexamethasone, the yellow fluorescence
intensities in Hela cells increased gradually which indicated
that the intracellular pH (especially these areas adjacent to the
cell nuclei) became more and more acidic. Figure 5 showed that
the addition of dexamethasone caused apoptosis of Hela cells
and time-dependent increasing of fluorescence intensity, which
in accordance with previous report on the apoptosis process and
the pH changes in HeLa cells.26 These results suggested that
intracellular acidifications might be possibly due to the release
of lysosomal proton in the apoptosis process. The increasing of
fluorescence intensity in HeLa cells induced by dexamethasone
was coincided with the results observed from RAW264.7 cells
induced by chloroquine.
Figure 2(A) 1H NMR spectra of CS-P and CS-P·HCl in (CD3)2SO-CDCl3 (4:1, v/v), (B)
Mechanism of pH response by protonation and deprotonation of CS-P
.
Testing of the selectivity, reversibility and photostability
Considering that nitrogen and oxygen could bind many metal
ions in solution, it was very important to determine whether
other ions were potential interferents. Upon addition of Na+,
K+, Mg2+, Ca2+, Ba2+, Mn2+, Cr3+, Ni2+, Cu2+, Pb2+, Zn2+, Fe3+
and Fe2+ to the 4 ꢀM CS-P solutions while keeping the
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substances can cause the leakage of protons out of lysosomal in
the form of protonated bases,32 while a parts of compounds
could decreased the pH value.15 However it was difficult to
determine the intracellular pH changes in a single cell. CS-P
proved a potential way to realize the detection of the cell pH
changes.
CS-P showed high pH sensitive ranged from pH 5.9 to pH
3.0, which was suitable for tracing the intracellular pH changes.
Furthermore, in order to determine the intracellular organelle
distribution of CS-P (2 ꢀM), we co-stained the MG63 cells
with LysoTracker® Red (0.1 ꢀM) (L7528, Invitrogen) or
MitoTracker® Deep Red, respectively. The results were shown
in Figure 6, the yellow fluorescence was observed in cytoplasm
(Figure 6A and 6D), but in the merged figure the CS-P did not
existed in lysosome (Figure 6C) or mitochondria (Figure 6F).
These results proved that CS-P was localized in cytoplasm.
C
A
B
D
F
E
Figure 4 Fluorescent intensities (yellow) were decreased distinctly along with pH
changes induced by chloroquine for mouse macrophage Raw 264.7.
Figure 6 Cellular Location experiment of CS-P. (A, D): Yellow emission for CS-P
;
(B) Red emission for LysoTracker® Red; (E) MitoTracker® Deep Red. (C) Merged A
and B. (F) Merged C and D.
Conclusions
In summary, a new coumarin derivative as pH sensitive probe
was designed and synthesized. CS-P exhibited high pH
sensitive ranged from pH 5.9 to pH 3.0 and its pKa = 4.55. In
vitro cell culture results showed that the fluorescence intensity
of CS-P could indicate the cellular pH changes distinctly.
Furthermore, CS-P was localized in cytoplasm after entered in
cells. These preliminary works demonstrated that CS-P was a
promising candidate for tracing cellular pH changes.
Acknowledgements
The authors acknowledge the financial support for this work by
Research Fund for the Doctoral Program of Higher Education
of China (20130181110089), the National Natural Science
Foundation of China (NSFC) (Grants No. 21372168) and
National Key Technology R&D Program (2012BAI42G01).
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/c000000x/
Figure 5 Fluorescent intensities of CS-P (yellow) were increased gradually along
with pH changes in HeLa cells induced by dexamethasome which was caused to
apoptosis status.
Notes and references
The pH value of cells would be changed in many cases,
such as cancer and Alzheimer's disease,33 weakly basic
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cDepartment of Biochemistry and Molecular Biology, West
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China School of Preclinical and Forensic Medicine, Sichuan 18 T. Myochin, K. Kiyose, K. Hanaoka, H. Kojima, T. Terai, T. Nagano, J
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*These two authors contributed equally to this work.
email : tanyf@scu.edu.cn (Y. Tan); jiangq@scu.edu.cn (Q.
Jiang)
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DOI: 10.1039/C4RA11347K
A novel coumarin derivative was synthesized and its application in live cell imaging
was demonstrated.