Synthesis of a new ratiometric emission Ca2+ indicator for in vivo bioimaging

Article abstract of DOI:10.1016/j.cclet.2012.12.016

A novel fluorescent calcium indicator with a 490/582 nm ratiometric emission has been designed and synthesized. The indicator exhibits a highly selective ratiometric emission response to Ca²⁺ over other metal cations and a large Stokes shift of 202 nm. Moreover, its practical cell imaging capability for intracellular Ca²⁺ in the resting- and dynamic-state has been demonstrated in human umbilical vein endothelial cells using a confocal laser scanning microscope.

Full text of DOI:10.1016/j.cclet.2012.12.016

Chinese Chemical Letters 24 (2013) 156–158
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Chinese Chemical Letters
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Original article
Synthesis of a new ratiometric emission Ca2+ indicator for in vivo bioimaging
Qiao-Ling Liu a,b, Meng Fan a, Wei Bian a, Shao-Min Shuang a, Chuan Dong a,
*
a Research Center of Environmental Science and Engineering, Shanxi University, Taiyuan 030006, China
b Department of Chemistry, Taiyuan Normal University, Taiyuan 030031, China
A R T I C L E I N F O
A B S T R A C T
Article history:
A novel fluorescent calcium indicator with a 490/582 nm ratiometric emission has been designed and
synthesized. The indicator exhibits a highly selective ratiometric emission response to Ca2+ over other
metal cations and a large Stokes shift of 202 nm. Moreover, its practical cell imaging capability for
intracellular Ca2+ in the resting- and dynamic-state has been demonstrated in human umbilical vein
endothelial cells using a confocal laser scanning microscope.
Received 4 November 2012
Received in revised form 29 November 2012
Accepted 5 December 2012
Available online 18 January 2013
ß 2012 Chuan Dong. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Keywords:
Ca2+ indicator
Ratiometric emission
Bioimaging
1. Introduction
form of BAPTA, intramolecular charge transfer (ICT) from the donor
to the acceptor will occur upon photon irradiation. Furthermore,
Calcium ion (Ca2+
)
as an almost universal intracellular
owing to the permeation of OXD–BAPTA–ester, it is possible to load
OXD–BAPTA–ester into living cells to monitor the intracellular
Ca2+ concentration in situ by confocal microscopic imaging.
Synthetic routes of OXD–BAPTA–ester and OXD–BAPTA were
depicted in Scheme 1 [5].
messenger plays diverse roles in intra- and intercellular signaling
[1], such as gene transcription, muscle contraction and cell
proliferation activities. Thus, tracking the dynamic changes of
intracellular Ca2+ concentration would certainly assist us in
recognizing many cellular processes and functions.
Fluorescent Ca2+ indicators as promising tools will benefit us to
enhance the understanding of the physiological roles of Ca2+
because they can provide spatial and temporal information about
the Ca2+ in cellular systems and in vivo [2]. Generally, the available
Ca2+ indicators include intensity- and ratiometric-based indica-
tors, and the latter is more desirable than the former in proper
detection of Ca2+. In the ratiometric-based Ca2+ indicators, only
Indo-1 and Indo-5F possess ratiometric fluorescence emission
behaviors toward the determination of Ca2+ concentration.
Unfortunately, Indo-1 and Indo-5F have to subject to UV excitation
2. Experimental
UV–vis absorption spectra were recorded on a Puxi TU-1901
UV-vis absorption spectrophotometer (China). 1H NMR and 13C-
NMR spectra were recorded on a Bruker AVANCE III 600 NMR
spectrometer (Germany). Elemental analysis was conducted on an
Elementar Vario EL Cube CHNOS analyzer (Germany). Mass
spectrum was acquired on a Bruker Autoflex matrix-assisted laser
desorption/ionization time of flight (MALDI-TOF) mass spectrom-
eter (Germany). Steady-state fluorescence measurements were
performed on an Edinburgh FLSP 920 spectrofluorometer (UK) at
21 ꢀ 1 8C. Synthetic routes of OXD–BAPTA–ester and OXD–BAPTA
were depicted in Scheme 1, and the compound OXD–BAPTA–ester
was fully characterized by 1H NMR, 13C NMR, mass spectrum and
elemental analyses. Melting point: 62–64 8C. The 1H NMR (600 MHz,
CDCl3): d 8.04 (d, 2H, J = 4.7 Hz, Ar–H), 7.82 (d, 2H, J = 6.2 Hz, Ar–H),
7.56 (d, 2H, J = 7.5 Hz, Ar–H), 7.38 (d, 1H, J = 5.8 Hz, Ar–H), 7.30 (d, 1H,
J = 5.6 Hz, Ar–H), 7.04 (d, 3H, J = 8.0 Hz, Ar–H), 6.91 (s, 1H, Ar–H), 6.80
(dd, 1H, J = 7.7 Hz, Ar–H), 6.66 (dd, 1H, J = 7.7 Hz and d, 1H, J = 6.1 Hz),
6.96 (d, 2H, J = 9.8 Hz, –CH55CH–), 4.60 (s, 4H, –CH2–CH2–), 4.31 (s,
8H, –CH2–), 4.12 (q, 10H, J = 7.5 Hz, –CH2–), and 1.31 (t, 15H,
J = 5.9 Hz, CH3–). The 13C NMR (150 MHz, CDCl3): d 171, 170, 164, 161,
145, 132, 128.6, 127, 126.8, 126.7, 125.5, 121, 118, 116, 114, 110, 67,
and their Kd are comparatively small (0.23 and 0.47
mmol/L,
respectively) so that their uses in cells and tissues are limited [3].
Herein, a novel fluorescent indicator based on ratiometric
emission measurements for Ca2+ is synthesized. We chose 2-(4-
ethoxyphenyl)-5-(4-methylphenyl)-1,3,4-oxadiazole as the fluor-
ophore and 1,2-bis(2-aminophenoxy)ethane-N,N,N0N0-tetraacetic
acid (BAPTA) group as the Ca2+ recognition site [4]. Due to the
electron deficient property of 1,3,4-oxadiazole (OXD), when it
binds to the electron rich groups of ethoxybenzene and the salt
* Corresponding author.
E-mail address: dc@sxu.edu.cn (C. Dong).
1001-8417/$ – see front matter ß 2012 Chuan Dong. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
http://dx.doi.org/10.1016/j.cclet.2012.12.016

Q.-L. Liu et al. / Chinese Chemical Letters 24 (2013) 156–158
157
OC2H5
O
H
N
O
(ii)
(i)
CH3
C2H5O
a)
N
H
CON HNH 2
N N
N N
O
(iii)
CH3
C2H5O
CH2Br
C2H5O
O
Compound 2
Compound 1
N(CH 2COOC 2H5)2
N(CH 2COOC 2H5)2
N(CH 2COOC 2H5)2
OCH 2CH2O
N(CH 2COOC 2H5)2
(iv)
OCH 2CH2O
b)
c)
CHO
BAPTA-tetraethyl ester
N N
Compound 3
N N
(v)
CH2Br--Ph3P+
CH2Br
C2H5O
C2H5O
O
Compound 2
O
N(CH 2COO-) 2
N(CH 2COOC 2H5)2
N(CH 2COO-) 2
N(CH 2COOC 2H5)2
OCH 2CH2O
OCH 2CH2O
CH
HC
CH
HC
(vii)
(vi)
N
N
N
N
O
O
OC2H5
OC2H5
Scheme 1. Synthesis of OXD–BAPTA. (a) (i) p-Toluoyl chloride, Py, reflux 3 h, 90%; (ii) POCl3, reflux 7 h, 60%; (iii) NBS, reflux 5 h, 90%; (b) (iv) POCl3, DMF, 54%; (c) (v) Ph3P; (vi)
NaH, compound 3, 60%; (vii) LiOH, 97%.
65.5, 61.2, 61.1, 14.7, and 14.0. MALDI-TOF MS: m/z: calcd. for
C
The stoichiometric ratio for the formation of OXD–BAPTA–Ca2+
complex was determined by the Job’s method and indicated a 1:1
ratio of OXD–BAPTA to Ca2+. The dissociation constant (Kd) of
48H54N4O12 :
878.3738; found: 901.5798 (M+Na+). Elemental
+
analysis calculated for C48H54N4O12: C 65.59, H 6.19, N 6.37, and O
21.84; found: C 65.78, H 6.20, N 6.39, and O, 21.89.
OXD–BAPTA–Ca2+ complex was determined as 0.56
0.080 mmol/L [6]. The fluorescence quantum yields (F) of OXD–BAPTA
mmol/L
ꢀ
3. Results and discussion
UV–vis spectra showed that the free OXD–BAPTA in HEPES
buffer solution (50 mmol/L, pH 7.2) exhibited a maximal absor-
bance at 380 nm. Upon addition of Ca2+ (0.0–11.1
mmol/L), the
absorbance at 380 nm decreased gradually, and showed a blue-
shift to 350 nm. When excited at 380 nm, OXD–BAPTA exhibited a
ꢀ
ꢁ
fl
max
maximal emission
l
at 582 nm, corresponding to the free
OXD–BAPTA (Fig. 1). On the addition of Ca2+ to OXD–BAPTA, the
lmflax shifted from 582 nm to 490 nm with a clear isoemissive point
at 518 nm, a large blue-shift of Dlem = 92 nm was observed,
indicating the formation of OXD–BAPTA–Ca2+ complex. Moreover,
the ratio of emission intensity (F490/F582) increased linearly with
the increasing Ca2+ concentrations; this result suggested that
OXD–BAPTA can be used as a good ratiometric indicator. Due to the
strong interaction between the acceptor and donor, a large Stokes
shift of 202 nm was found, which implied the spectral interference
from the excitation light beam and autofluorescence can be
minimized. Accordingly, the fluorescence color changed from fade
orange to bright green under the irradiation of an UV lamp (inset of
Fig. 1).
Fig. 1. Fluorescence emission spectra of 1.0
mmol/L OXD–BAPTA in 50 mmol/L
HEPES (pH 7.2) containing 100 mmol/L KCl and 10 mmol/L EGTA in the presence of
various concentrations of Ca2+ ions (1–13): 0.00, 0.026, 0.038, 0.098, 0.15, 0.227,
0.341, 0.582, 0.906, 2.04, 3.56, 5.45, and 11.1
mmol/L at excitation wavelength of
380 nm. The inset showed the fluorescence emission images of OXD–BAPTA before
(left) and after (right) addition of Ca2+ under an UV lamp irradiation.

158
Q.-L. Liu et al. / Chinese Chemical Letters 24 (2013) 156–158
Fig. 2. Ratiometric fluorescence intensity (F490/F582) of 1.0
mmol/L OXD–BAPTA in
the presence of 11.1
Ba2+, Ni2+, Sr2+, Hg2+, Pb2+ (black bars) followed by adding to 11.1
m m ,
mol/L Ca2+, 150 mol/L Mg2+, Zn2+, Co2+, Mn2+, Fe2+, Cu2+
Fig. 3. Confocal fluorescence images of intracellular Ca2+ in living HUVEC. The
excitation wavelength was 458 nm (a) and (b) OXD–BAPTA loaded HUVEC,
observing emission wavelengths at 560ꢁ600 nm and 470ꢁ510 nm, respectively. (c)
and (d) Penicillin G sodium salt (30 U/mL) was added to the OXD–BAPTA stained
HUVEC. The images were captured after 180 s at emission wavelengths of
560ꢁ600 nm and 470ꢁ510 nm, respectively.
m
mol/L Ca2+ (red
bars) in 50 mmol/L HEPES (pH 7.2) containing 100 mmol/L KCl. The excitation
wavelength was 380 nm. (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of the article.)
and OXD–BAPTA–Ca2+ complex were determined as 0.022 and 0.017 at
excitation wavelength of 380 nm, respectively. Quinine bisulfate in
0.050 mol/L H2SO4 (F of 0.546) was used as the fluorescence standard.
The effects of various metal ions on OXD–BAPTA were also
investigated under physiological conditions (Fig. 2). Ca2+ exhibits
the largest effect on the ratiometric fluorescence intensity
for Ca2+ in the presence of other competing cations with a large
Stokes shift of 202 nm and an obvious color change. Dual emissions
and the cell-permeable nature of OXD–BAPTA–ester make it
possible to study cellular Ca2+ in living HUVEC by confocal laser
scanning microscope. These special traits indicate that OXD–
BAPTA is an excellent Ca2+ indicator for in vivo bioimaging.
(490 nm/582 nm) of OXD–BAPTA, on which Mg2+, Zn2+, Co2+
,
Mn2+, Fe2+, Cu2+, Ba2+, Ni2+, Sr2+, Hg2+, Pb2+ have a slight effect.
Thus, OXD–BAPTA can selectively detect Ca2+ over other metal
cations.
Further studies were performed to explore the potential use of
OXD–BAPTA for the detection of intracellular Ca2+ in living cells.
Herein, OXD–BAPTA–ester was employed for its higher cell
permeability. Human umbilical vein endothelial cells (HUVEC)
Acknowledgment
This work was supported by the National Natural Science
Foundation of China (Nos. 21175086 and 21175087).
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In conclusion, we have successfully developed the ratiometric
fluorescent indicator OXD–BAPTA for Ca2+ based on the ICT
mechanism. OXD–BAPTA exhibits selective ratiometric detection