A novel ratiometric emission probe for Ca2+ in living cells

Article abstract of DOI:10.1039/c2ob26888d

A ratiometric fluorescent probe for Ca²⁺ based on 1,3,4-oxadiazole derivative has been designed and developed. The probe exhibits a large Stokes shift of 202 nm and a highly selective ratiometric emission response (490/582 nm) to Ca²⁺ over other metal cations. Additionally, the probe can readily reveal the changes of intracellular Ca²⁺ concentration in living human umbilical vein endothelial cells. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c2ob26888d

Organic &
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A novel ratiometric emission probe for Ca2+ in
living cells†
Cite this: Org. Biomol. Chem., 2013, 11,
503
Qiaoling Liu,a,b Wei Bian,a Heping Shi,a Li Fan,a Shaomin Shuang,a Chuan Dong*a
and Martin M. F. Choi*c
Received 25th September 2012,
Accepted 6th November 2012
A ratiometric fluorescent probe for Ca2+ based on 1,3,4-oxadiazole derivative has been designed and
developed. The probe exhibits a large Stokes shift of 202 nm and a highly selective ratiometric emission
response (490/582 nm) to Ca2+ over other metal cations. Additionally, the probe can readily reveal the
changes of intracellular Ca2+ concentration in living human umbilical vein endothelial cells.
DOI: 10.1039/c2ob26888d
www.rsc.org/obc
fast transients, the probe with small Kd could saturate quickly
and results in inaccurate measurement. On the other hand, if
Introduction
Calcium ion (Ca2+) as a second intracellular messenger plays the Kd is too large, Ca2+ measurement will be insensitive.4 So
multiple crucial roles in intra- and intercellular signaling. the chosen Ca2+ probe with an appropriate value of Kd is of
Through the temporal and spatial fluctuations of Ca2+ concen- great importance for accurate intracellular Ca2+ measurement.
trations, many different cellular processes including fertiliza-
In addition, in view of intensity- and ratiometric-based Ca2+
tion, proliferation, metabolism, contraction and apoptosis can probes, the latter is more desirable than the former in proper
be regulated.1 The total calcium content in resting cells is gen- detection of Ca2+ since ratiometric probes allow for internal
erally 1–7 mM, but most of the cellular calcium (>99.9%) is calibration of Ca2+ and obviate the experimental corrections of
stored in organelles such as mitochondria and endoplasmic photobleaching, sample thickness variability and differential
reticulum. The free Ca2+ is only around 100 nM. However, loading of probe within the cell.5 To date, only a few ratio-
during various cellular functions, the free Ca2+ concentration metric Ca2+ probes, including Fura-2, Indo-1 and Indo-5F, are
can be regulated by the release of Ca2+ from the intracellular commercially available and they have been widely utilized to
Ca2+-storing organelles and the concentration sometimes goes monitor the changes of intracellular Ca2+ and assisted us in
up 10–100 folds.2 Thus, tracking the dynamic changes of intra- recognizing some diseases such as hyperparathyroidism,
cellular Ca2+ concentration and understanding the signal Ca2+ tumor metastasis and renal failure.6 It is noteworthy that these
pathways would certainly help us recognize many cellular pro- ratiometric Ca2+ probes all adopt 1,2-bis(2-aminophenoxy)
cesses and functions.
ethane-N,N,N′,N′-tetraacetic acid (BAPTA) structure for selec-
Up to now, a successful approach for intracellular Ca2+ tively binding Ca2+.7 This type of Ca2+ probe ensures good sen-
measurement is to introduce the optically sensitive Ca2+ sitivity and selectivity for Ca2+ over other physiological metal
probes into living cells, combining with digital imaging ions. To our knowledge, in this kind of ratiometric Ca2+
microscopy. This technique possesses the advantages of high probes, only Indo-1 and Indo-5F can perform emission ratio
sensitivity, good selectivity, fast response time, and in situ measurements as a function of Ca2+ concentration. Unfortu-
observation.3 A Ca2+ probe works best if its dissociation con- nately, Indo-1 and Indo-5F require UV excitation which will
stant (Kd) is close to the intracellular Ca2+ concentration. inevitably lead to photobleaching, high scattering background
When the calcium concentration is higher or is involved in and autofluorescence interference.8 Moreover, their Kd are rela-
tively small (0.23 and 0.47 μM, respectively) so that their appli-
cations in cells and tissues are quite limited.
aResearch Center of Environmental Science and Engineering, and School of
In this paper, we report the development of a novel Ca2+
Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006,
probe based on ratiometric emission measurement. Our mo-
P. R. China. E-mail: dc@sxu.edu.cn; Fax: +86-0351-7018613; Tel: +86-0351-7018613
bDepartment of Chemistry, Taiyuan Normal University, Taiyuan 030031, P. R. China
cDepartment of Chemistry, Hong Kong Baptist University, 224 Waterloo Road,
Kowloon Tong, Hong Kong SAR, P. R. China. E-mail: mfchoi@hkbu.edu.hk;
lecular design strategy is to incorporate 2-(4-ethoxyphenyl)-5-
(4-methylphenyl)-1,3,4-oxadiazole as the fluorophore and BAPTA
group as the Ca2+ recognition site. To our knowledge, 1,3,4-
oxadiazole (OXD) derivative based Ca2+ probe has not been
reported before. Owing to the electron-deficient property of
OXD, when it conjugates to the electron-rich groups of
Fax: +852-34117348; Tel: +852-34117839
†Electronic supplementary information (ESI) available: Determinations of dis-
sociation constants and quantum yields, Supplementary figures, NMR spectra
and mass spectrum. See DOI: 10.1039/c2ob26888d
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ethoxybenzene and salt form of BAPTA, intramolecular charge
transfer (ICT) from the donor to the acceptor will proceed
Results and discussion
upon photon irradiation. This push–pull conjugated molecule Synthetic routes of OXD–BAPTA–ester and OXD–BAPTA are
will exhibit a large Stokes shift attributing to its large π-elec- depicted in Scheme 2, compound 1 (2-(4-ethoxyphenyl)-5-
tron conjugation system. In addition, since there is an overlap (4-methylphenyl)-1,3,4-oxadiazole) and compound 2 (2-[(4-bro-
of the excitation spectrum of OXD fluorophore (260–360 nm) momethyl)phenyl]-5-(4-ethoxyphenyl)-1,3,4-oxadiazole)
were
with that of BAPTA (230–320 nm), the same excitation wave- prepared according to previous reports with slight modifi-
length could be applied to trigger the emissions of OXD fluoro- cations.9 Compound 3 (5-formyl-BAPTA-tetraethyl ester) was
phore and BAPTA. It is envisaged that Ca2+ coordination to obtained by following the Grynkiewicz et al. method.10 Com-
BAPTA will alter the emission ratio of OXD fluorophore to pound 2 and 3 were coupled by the Wittig reaction under N2
BAPTA, offering ratiometric Ca2+-sensing capability. When atmosphere and subsequently purified by column chromato-
BAPTA binds with a Ca2+ ion, its electron-donating property graphy to obtain the yellow solid OXD–BAPTA–ester in 60%
will be restricted with a concomittant reduction of π-electron yield. OXD–BAPTA was obtained by base hydrolysis of OXD–
conjugation and, as a result, both absorption and emission BAPTA–ester in aqueous lithium hydroxide, and the OXD–
spectra are blue-shifted, which will provide potential for Ca2+ BAPTA–ester was fully characterized by 1H NMR, 13C NMR,
detection. Furthermore, since OXD–BAPTA–ester permeates mass spectrum and elemental analyses (ESI†).
the membrane easily, OXD–BAPTA–ester loaded cells will
The absorption spectral change of OXD–BAPTA in HEPES
provide an opportunity to observe the intracellular Ca2+ signal buffer solution (50 mM, pH 7.2) in the presence of various con-
in situ by confocal microscopic imaging. The structures and centrations of Ca2+ ion are displayed in Fig. 1. Free OXD–
color changes of OXD–BAPTA and its Ca2+-complex are BAPTA shows two strong absorption bands centered at 305 nm
depicted in Scheme 1.
(ε = 3.43 × 104 M−1 cm−1) and 380 nm (ε = 4.46 × 104 M−1
cm−1), assigned to the π–π* transition and ICT bands, respect-
ively. On the addition of Ca2+ (0.0–11.1 μM) to the 10.0 μM
OXD–BAPTA solution, the bands at 305 and 380 nm decrease
gradually and the ICT band exhibits a large hypsochromic
shift to 350 nm with the solution changing color from orange
to faint greenish yellow (Inset of Fig. 1).
Fig. 2 depicts the fluorescence emission spectral changes of
OXD–BAPTA after the addition of Ca2+ ion under physiological
conditions. At an excitation wavelength of 380 nm (Fig. 2a), a
strong emission band with a fluorescence emission maximum
fl
(λ ) of 582 nm is observed, corresponding to the free OXD–
max
BAPTA. On addition of Ca2+ to OXDA–BAPTA, the λ
shifts
fl
max
from 582 to 490 nm. An isoemissive point of 518 nm is clearly
found, indicating the formation of OXD–BAPTA–Ca2+ complex.
A similar result is obtained at the excitation wavelength of
Scheme 1 The structures of OXD–BAPTA and its Ca2+-complex with color
changes.
Scheme 2 Synthetic procedures of OXD–BAPTA. Conditions: (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%.
504 | Org. Biomol. Chem., 2013, 11, 503–508
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Table 1 Spectra properties of ratiometric emission Ca2+ probes
Probe
Ex (nm)
Em (nm)
Stokes shift (nm)
Kd (μM)
Indo-1a
346
344
380
401/475
398/471
490/582
129
127
202
0.23
0.47
0.56 0.08
Indo-5Fa
OXD–BAPTAb
Spectra data and Kd values measured in 100 mM KCl, 10 mM EGTA
and (a) 10 mM MOPS or (b) 50 mM HEPES at pH 7.20.
Fig. 1 Absorption spectra of 10.0 μM OXD–BAPTA in 50 mM HEPES (pH 7.2)
containing 100 mM KCl and 10 mM EGTA in the presence of various concen-
trations of Ca2+ ions (1–9: 0.00, 0.038, 0.098, 0.227, 0.341, 0.582, 0.906, 2.04,
and 11.1 μM). EGTA stands for ethylene glycol bis(β-aminoethyl ether)-N,N,N’,N’-
tetraacetic acid. The inset shows the images of OXD–BAPTA before (left) and
after (right) addition of Ca2+
.
Fig. 3 (a) The fluorescence spectra of 1.0 μM OXD–BAPTA in the presence of
11.1 μM Ca2+, 150 μM Mg2+, Zn2+, Co2+, Mn2+, Fe2+, Cu2+, Ba2+, Ni2+, Sr2+, Hg2+
,
Pb2+, Na+, K+ and Al3+ in 50 mM HEPES (pH 7.2) containing 100 mM KCl. (b)
Relative fluorescence intensity of 1.0 μM OXD–BAPTA in the presence of 150 μM
Mg2+, Zn2+, Co2+, Mn2+, Fe2+, Cu2+, Na+ and K+ (orange bars) followed by
addition of 11.1 μM Ca2+ (olive bars) in 50 mM HEPES (pH 7.2) containing
100 mM KCl. Fo and F are the fluorescence intensities of OXD–BAPTA in the
absence and presence of metal ions, respectively. The excitation wavelength is
380 nm.
Fig. 2 (a) Fluorescence emission spectra of 1.0 μM OXD–BAPTA in 50 mM
HEPES (pH 7.2) containing 100 mM KCl and 10 mM 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 μM at excitation wave-
lengths of 380 nm. The inset shows the fluorescence emission images of OXD–
BAPTA before (left) and after (right) addition of Ca2+ under UV lamp irradiation.
(b) Ratiometric calibration curve F490/F582 as a function of Ca2+ concentration.
The stoichiometric ratio for the formation of OXD–BAPTA–
Ca2+ complex was determined by the Job’s method and the
result indicates the formation of OXD–BAPTA–Ca2+ complex
with a 1 : 1 ratio of OXD–BAPTA to Ca2+ (Fig. 1S, ESI†). The dis-
sociation constant (Kd) of OXD–BAPTA–Ca2+ complex was
determined as 0.56 0.080 μM (Fig. 2S, ESI†), suggesting that
the dissociation constant is relatively small, but larger than
350 nm. Accordingly, the fluorescence color changes from pale
red to bright green under irradiation of an UV lamp (Inset of
Fig. 2a). The ratio of fluorescence intensity F490/F582 also that of Indo-1 and Indo-5F (Table 1). Thus, OXD–BAPTA could
bind to Ca2+ with good stability. The spectral response of
OXD–BAPTA toward Mg2+ was also studied (Fig. 3S, ESI†). The
emission spectra show that OXD–BAPTA for Mg2+ is only an
intensity-based probe and the OXD–BAPTA–Mg2+ complex is
formed the 1 : 1 ratio of OXD–BAPTA to Mg2+ which is deter-
changes linearly with the concentration of Ca2+ (Fig. 2b), this
result suggests that OXD–BAPTA can be used as a good ratio-
metric probe.
The absorption and emission spectral properties illustrate
that Ca2+ ion binds to the BAPTA moiety of OXD–BAPTA so as
to weaken the ability of BAPTA to participate in the π-electron mined by the Hill plot. The Kd for OXD–BAPTA–Mg2+ is 4.35
0.075 mM, suggesting the affinity of OXD–BAPTA to Mg2+ is
conjugation of the molecule. Thus, the π-electron density in
the conjugated molecule decreases, resulting in reduction of
much smaller than that of Ca2+.
ICT. On the whole, both absorption and emission bands are
blue-shifted. The spectrofluorometric data also provide us
valuable information for exploring the potential application of
The fluorescence spectra of OXD–BAPTA with various metal
cations are described in Fig. 3a. Among these metal ions, Ca2+
exhibits the largest effect on the fluorescence intensity of
OXD–BAPTA for determination of intracellular Ca2+ since there OXD–BAPTA. Fig. 3b displays the normalized relative response
of OXD–BAPTA to the physiological metal ions. Mn2+ and Co2+
is a large Stokes shift of 202 nm between the excitation
(380 nm) and emission (582 nm) peaks. This can minimize
show moderate effect while Fe2+, Mg2+, Zn2+, Cu2+, Na+ and K+
spectral interference from the excitation light beam and auto- have slight effect on the fluorescence intensity of OXD–BAPTA.
Fortunately, the intracellular concentrations of free Mn2+ and
fluorescence. Moreover,
a
large spectral shift (92 nm)
Co2+ ions are negligible. In addition, OXD–BAPTA shows good
fl
between the two λ
(490 and 582 nm) and their changes in
max
emission intensity result in a huge ratiometric value (Fig. 2a response to Ca2+ when both Ca2+ and the physiological metal
ions are co-existed in the HEPES buffer solution, indicating
and 2b).
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that OXD–BAPTA displays excellent selectivity for Ca2+ over
other physiological cations so that OXD–BAPTA could be
employed to measure the intracellular free Ca2+. Furthermore,
the pH effect on OXD–BAPTA in the biologically relevant pH
range determines that the optimal condition is pH 7.0–7.5
(Fig. 4S, ESI†).
The fluorescence quantum yields (Φ) of OXD–BAPTA and
OXD–BAPTA–Ca2+ complex were determined as 0.007 and
0.010 at an excitation wavelength of 350 nm; 0.022 and 0.017
at excitation wavelength of 380 nm, respectively. Quinine bisul-
fate in 0.050 M H2SO4 (Φ of 0.546) was used as the fluor-
escence standard (ESI†). The small Φ is attributed to the polar
solvent of water. Polar solvent–solute interactions lead to a
reduced coplanar effect, more efficient nonradiative deactiva-
tion and finally lead to a drastically reduced brightness.11 For
the OXD–BAPTA–Ca2+ complex, Ca2+-binding induces
a
reduction of ICT which in turn results in a smaller Φ. The flu-
orescence lifetime (τ) for OXD–BAPTA and OXD–BAPTA–Ca2+
were determined from time-resolved fluorescence spectroscopy
as 1.52 and 1.51 ns at excitation wavelength of 350 nm;
1.16 and 1.15 ns at excitation wavelength of 380 nm,
respectively.
To explore the potential application of OXD–BAPTA in
living cell imaging and the dynamic change of intracellular
Ca2+ signal, OXD–BAPTA–ester was employed for cell per-
meability. Human umbilical vein endothelial cells (HUVEC)
were incubated with OXD–BAPTA–ester (5.0 μM) for 40 min at
37 °C, and then the ester was converted to the active form of
OXD–BAPTA inside the cells by intracellular esterase, which
ensured the Ca2+ signal could be deciphered in the cytosol.
Prior to imaging, the medium was removed, and the OXD–
BAPTA stained HUVEC were washed with HEPES three times.12
Confocal fluorescent imaging was performed at an excitation
wavelength of 458 nm since 380 nm laser was not available for
the confocal microscope, the emission wavelengths were cen-
tered at 580 20 nm and 490 20 nm for the fluorescent pro-
perties of OXD–BAPTA and its Ca2+-complex. Fig. 4a and 4b
depict images of the stained cells with bright red and faint
green fluorescence at the above-mentioned emission wave-
lengths, indicating a lower free Ca2+ concentration in cytosol
as the resting levels of [Ca2+]i. Then these OXD–BAPTA stained
cells were given to penicillin G sodium salt (30 U mL−1), the
cell images were captured again after 180 s as depicted in
Fig. 4c and 4d. The red fluorescence drops dramatically and
the green fluorescence was enhanced, inferring the release of
[Ca2+]i from the intracellular Ca2+-storing organelles after
administration of penicillin G sodium salt. Fig. 4e records the
changes of the fluorescence intensities at the marked circle
areas 1 and 2 for 180 s at the above-mentioned emission wave-
lengths. Fig. 4f exhibits the ratiometric change F490/F582 for
180 s. These results indicate that the intracellular [Ca2+]i
can be determined in real-time after the living cells have
Fig. 4 Confocal fluorescence images of intracellular Ca2+ in living HUVEC. The
excitation wavelength is 458 nm, the emission wavelengths are centered at
580 20 nm and 490 20 nm (20 × objective lens). (a), (b) HUVEC are incu-
bated with 5.0 μM OXD–BAPTA for 40 min at 37 °C, observing emission wave-
lengths at 580
20 nm and 490
20 nm, respectively. (c), (d) Penicillin G
sodium salt (30 U mL−1) is added to the OXD–BAPTA stained HUVEC. The
images are captured after 180 s at emission wavelengths of 580 20 nm and
490
20 nm, respectively. (e) Fluorescence intensities are recorded at the
marked circle areas 1 and 2 from (a) to (c) and (b) to (d) for 180 s at emission
wavelengths of 580 20 nm and 490 20 nm, respectively. (f) Ratiometric flu-
orescence change F490/F582 for 180 s.
Conclusions
In summary, we have developed an emission ratiometric probe
(OXD–BAPTA) for detecting intracellular [Ca2+]i. The main attri-
butes of this probe are that it displays high sensitivity and
selectivity for Ca2+ over other metal ions, a large Stokes shift of
202 nm and enables ratiometric emission measurement with
an obvious color change. Moreover, OXD–BAPTA can track the
[Ca2+]i signal in real-time in living HUVEC by microscopic
imaging. These special traits indicate that OXD–BAPTA is an
excellent probe for intracellular Ca2+ signal.
Experimental section
Materials and characterization
been exposed to the irritation of drugs. In essence, OXD– All chemicals were purchased from Shanghai Aladdin Reagent
BAPTA is obviously capable of visualizing the intracellular Co., Ltd. Organic solvents (AR grade) were purchased from
[Ca2+]i waves in living cells under a confocal laser scanning commercial suppliers without further purification before use.
microscope.
Deionized water was obtained from a Milli-Q water purification
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system (Millipore). 50 mM HEPES buffer solution (pH 7.2) con- ethyl acetate. The combined organic extracts were concentrated
taining 100 mM KCl and 10 mM EGTA was used to prepare the under reduced pressure to obtain compound 1 (1.47 g, 60%
OXD–BAPTA solutions. The stock solutions of 1.0 mM OXD– yield) as faint yellow crystalline powder. Melting point:
1
BAPTA–ester were prepared by dissolving OXD–BAPTA–ester in 129–132 °C. H NMR (600 MHz, CDCl3): δ 8.00 (d, Ar–H, 2H),
dimethyl sulfoxide and kept at −18 °C. 0.20 M stock solutions 7.91 (d, Ar–H, 2H), 7.30 (d, Ar–H, 2H), 7.05 (d, Ar–H, 2H), 4.10
of Ca2+, Mg2+, Zn2+, Co2+, Mn2+, Fe2+, Cu2+, Ba2+, Ni2+, Sr2+, (q, –CH2–, 2H), 2.34 (s, CH3–, 3H), and 1.32 (t, CH3–, 3H).
Hg2+, Pb2+, Na+, K+ and Al3+ were prepared from their chloride
Synthesis of 2-[(4-bromomethyl)phenyl]-5-(4-ethoxyphenyl)-
1,3,4-oxadiazole (2)
salts. 0.20 M Fe2+ solution and 1500 U mL−1 penicillin G
sodium salt were freshly prepared just before experiments. UV-
vis absorption spectra were recorded on a Puxi TU-1901 UV-vis Compound 1 (1.47 g, 5.0 mmol), N-bromosuccinimide (0.95 g,
absorption spectrophotometer (China). 1H NMR spectra and 5.0 mmol) and 0.015 g benzoyl peroxide were dissolved in
13C NMR were recorded on a Bruker AVANCE III 600 NMR spec- 12 mL dry benzene. The mixture solution was stirred and
trometer (Germany). Elemental analysis was conducted on an heated to reflux for 5 h. Benzene was removed on a rotary evap-
Elementar Vario EL Cube CHNOS analyzer (Germany). Mass orator and the precipitate was recrystallized from hot ethanol
spectrum was acquired on a Bruker Autoflex matrix-assisted to obtain 2-[(4-bromomethyl)phenyl]-5-(4-ethoxyphenyl)-1,3,4-
laser desorption/ionization time of flight (MALDI-TOF) mass oxadiazole (2, 1.70 g, 90% yield) as yellow crystalline powder.
1
spectrometer (Germany). Steady-state fluorescence measure- Melting point: 104–108 °C. H NMR (600 MHz, CDCl3): δ 8.00
ments were performed on an Edinburgh FLSP 920 spectrofluo- (d, Ar–H, 2H), 7.95 (d, Ar–H, 2H), 7.30 (d, Ar–H, 2H), 7.05
rometer (UK) at 21 1 °C.
(d, Ar–H, 2H), 4.10 (q, –CH2–, 2H), 4.60 (s, –CH2–, 2H), and
1.32 (t, CH3–, 3H).
Cell culture and imaging
Synthesis of 5-formyl-BAPTA-tetraethyl ester (3)
Human umbilical vein endothelial cells (HUVEC) were cul-
tured in DEME supplemented with 10% FCS (Invitrogen). One 5-Formyl-BAPTA-tetraethyl ester (3) was synthesized following
day before cell imaging, HUVEC were seeded on cover slips. the Grynkiewicz et al. method.10 16 mL phosphorus oxychlor-
The next day, HUVEC were incubated with 5.0 μM OXD– ide was added dropwise to the solution of BAPTA–tetraethyl
BAPTA–ester for 40 min at 37 °C under 5% CO2, washed with ester (6.0 g, 10.2 mmol) in 30 mL dry DMF and 21 mL dry pyri-
HEPES buffer three times and then subjected to cell imaging dine at 0 °C. The reaction mixture was stirred for 30 min at
under an Olympus FluoView 1000 confocal laser scanning room temperature, followed for 1 h at 70 °C and overnight at
microscope using an objective lens (20×) and an excitation room temperature. Then 100 mL chloroform was added to
light beam of 458 nm. The fluorescence intensity was extract twice the product. This extracted solution was washed
recorded. Next, 30 U mL−1 penicillin G sodium salt was added with ice water to neutral and dried over Na2SO4. The filtrate
to these OXD–BAPTA–ester incubated cells and simultaneously was removed under reduced pressure and concentrated under
recorded the change of intracellular Ca2+ signal ([Ca2+]i) within reduced pressure. This concentrated solution was purified by
180 s. For all imaging, the microscope settings such as bright- column chromatography using silica gel as the stationary
ness, contrast, and exposure time were held constant to phase and ether/ethyl acetate (1 : 1) as the eluent to obtain
1
compare the changes in the fluorescent intensities of incu- 3.41 g (54% yield) white powder. H NMR (600 MHz, CDCl3): δ
bated cells with or without penicillin G sodium salt.
9.63 (s, –CHO, 1H), 7.51 (d, Ar–H, 1H), 7.30 (d, Ar–H, 1H), 6.88
(dd, Ar–H, 2H), 6.65 (d, Ar–H, 2H), 6.38 (d, Ar–H, 1H), 4.58(s,
–CH2–CH2–, 4H), 4.32 (s, –CH2–, 8H), 4.12 (q, –CH2–, 8H), and
1.32 (t, CH3-, 12H).
Synthesis of 2-(4-ethoxyphenyl)-5-(4-methyl phenyl)-1,3,4-
oxadiazole (1)
4-Methylbenzyol chloride was initially prepared by reacting
4-methylbenzoic acid (1.81 g, 13.3 mmol) with 2 mL thionyl
Synthesis of OXD–BAPTA–ester
chloride under reflux for 3 h. The reaction mixture was concen- Compound 2 (1.70 g, 4.73 mmol) and triphenylphosphine
trated under reduced pressure to give the crude acid chloride (1.36 g, 4.89 mmol) were added to 25 ml dry benzene and the
yellow solution. It was then gradually added to 10 mL CHCl3 mixture was heated under reflux for 4 h. After cooling to room
containing 4-ethoxybenzhydrazide (2.18 g, 12.1 mmol) and temperature, the reaction mixture was filtered to obtain the
1.1 mL pyridine. The mixture was stirred under reflux for 3 h white solid phosphorane ylide and dried in vacuum (2.9 g,
and concentrated under reduced pressure. The residue was 4.67 mmol). Sodium hydride (0.16 g, 4.0 mmol) was put into
washed with water and recrystallized from hot ethanol; a white 5 mL of petroleum ether under N2 atmosphere and stirred for
crystalline powder was obtained (3.24 g, 90%) and dried in 20 min. The petroleum ether was discarded and replaced with
vacuum. The dried white crystalline powder was heated in 15 mL dried CH2Cl2. Phosphorane ylide (2.0 g, 3.24 mmol) was
10 mL POCl3 under reflux for 7 h. The solution was concen- added and stirred at room temperature for 1 h. Compound 3
trated under reduced pressure to give the crude solid product. (2.0 g, 3.24 mmol) was loaded into the mixture and stirred at
After cooling to room temperature, the precipitate was washed room temperature for 4 h. The mixture was concentrated and
with ice water. An aqueous solution of sodium acetate (50% purified by column chromatography using silica gel as the
w/w) was added and the precipitate was extracted twice with stationary phase and n-hexane/ethyl acetate (1 : 1) as the eluent
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to obtain the yellow solid OXD–BAPTA–ester (1.7 g, 60% yield).
Melting point: 62–64 °C. H NMR (600 MHz, CDCl3): δ 8.04 (d,
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1
Ar–H, 2H), 7.82 (d, Ar–H, 2H), 7.56 (d, Ar–H, 2H), 7.38 (d, Ar–
H, H), 7.30 (d, Ar–H, H), 7.04 (d, Ar–H, 3H), 6.91 (s, Ar–H, 1H),
6.80 (dd, Ar–H, 1H), 6.66 (dd, 1H and d, 1H), 6.96 (d,
–CHvCH–, 2H), 4.60 (s, –CH2–CH2–, 4H), 4.31 (s, –CH2–, 8H),
4.12(q, –CH2–.10H), and 1.31 (t, CH3–, 15H). 13C NMR
(600 MHz, CDCl3): δ 171, 170, 164, 161, 145, 132, 128.6, 127,
126.8, 126.7, 125.5, 121, 118, 116, 114, 110, 67, 65.5, 61.2, 61.1,
14.7 and 14.0. MALDI–TOF MS: m/z calculated for
C48H54N4O12+: 878; 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%.
Synthesis of OXD–BAPTA
OXD–BAPTA was obtained by hydrolyzing OXD–BAPTA–ester in
0.50 M LiOH solution in an ice bath for 3 h. After hydrolysis,
the pH was adjusted to 2–3 by dropwise addition of 1.0 M HCl
with stirring. A pale yellow precipitate was collected by fil-
tration and washed with 3 mL ice-water three times. The pre-
cipitate was put into the equivalent LiOH solution and stirred
at 0 °C. Finally, the purified OXD–BAPTA product was dried in
vacuum and kept at −18 °C until further use.
Acknowledgements
This work was supported by grants from the National Natural
Science Foundation of China (21175086 and 21175087), and
the Hundred Talent Programme of Shanxi Province, Shanxi
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