Fluorescent sensors based on controllable conformational change for discrimination of Zn2+ over Cd2+

Article abstract of DOI:10.1016/j.tet.2012.04.090

A new coumarin-derived imine IC1 was designed for highly selective sensing of Zn²⁺ over Cd²⁺ ions in aqueous solution based on a controllable CN isomerization mechanism. Methyl as a fine controllable unit was incorporated into IC1 to tune the conformational change of imine and thus significantly improve its selectivity to Zn²⁺ compared with methyl-free imine IC2. Sensor IC1 was also utilized to image intracellular Zn²⁺ ions in HepG2 cells with a good performance.

Full text of DOI:10.1016/j.tet.2012.04.090

Tetrahedron 68 (2012) 5458e5463
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Fluorescent sensors based on controllable conformational change
for discrimination of Zn2þ over Cd2þ
*
Jiasheng Wu, Ruilong Sheng, Weimin Liu, Pengfei Wang , Hongyan Zhang, Jingjin Ma
Key Laboratory of Photochemical Conversion and Optoelectronic Materials, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100190, PR China
a r t i c l e i n f o
a b s t r a c t
A new coumarin-derived imine IC1 was designed for highly selective sensing of Zn2þ over Cd2þ ions in
aqueous solution based on a controllable C]N isomerization mechanism. Methyl as a fine controllable
unit was incorporated into IC1 to tune the conformational change of imine and thus significantly im-
prove its selectivity to Zn2þ compared with methyl-free imine IC2. Sensor IC1 was also utilized to image
intracellular Zn2þ ions in HepG2 cells with a good performance.
Article history:
Received 26 November 2011
Received in revised form 17 April 2012
Accepted 24 April 2012
Available online 2 May 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Controllable C]N isomerization
Conformational change
Recognition of Zn2þ from Cd2þ
Zn2þ imaging
1. Introduction
usually non-fluorescent, while their analogues with cyclic C]N
double bonds are significantly fluorescent.8
Zinc element, as a cofactor existing in a large number of proteins
and enzymes, plays important roles in biological system.1,2 It is
responsible for neurological disorders such as Alzheimer’s disease,
Parkinson’s disease, as well as insulin secretion and apoptosis.3
Considerable interest has been focused on the development of
specific detection of zinc ions.4 So far, a number of fluorescent
sensors for Zn2þ have been developed generally based on photo-
induced electron transfer (PET)5 and intramolecular charge trans-
fer (ICT) mechanisms.6 However, most of these sensors have dis-
played the inter-crossing disturbance from Cd2þ because zinc and
cadmium are located in the same group of the Periodic Table and
can result in very similar photophysical changes of sensors.4 Con-
sequently, it is still a challenge to design new fluorescent sensors to
Following this mechanism, herein, we demonstrated a new
‘turn-on’ fluorescent sensor (IC1) to discriminate Zn2þ over Cd2þ
ions based on a C]N isomerization via a controllable conforma-
tional change (Fig. 1b). IC1 was designed as a Zn2þ-selective fluo-
rescent sensor on the basis of the consideration as follows, (i) the
methyl group as a minor control unit has been incorporated into
the imine in IC1 to tune its sensing selectivity for metal ions; (ii)
a methyl-free analogue IC2 was synthesized to study its selectivity
for comparison; (iii) the carbonyl in coumarin and the pyridyl
Isomerization
Isomerization inhibited
Strongly fluorescent
Non-fluorescencet
clearly discriminate Zn2þ from Cd2þ
.
N
N
(a)
N
In the course of our continuing exploration of fluorescent sen-
sors, we have firstly developed an imine-derived sensor (IC) for
Zn2þ ions based on C]N isomerization as a new sensing mecha-
nism (Fig.1a).7 In that case, the imine with an unfixed C]N struc-
ture is non-fluorescent because the C]N isomerization is the
predominant decay process in the excited states. In contrast, the
complexation with Zn2þ ions restricts the rotation of C]N bond
and results in the suppression of C]N isomerization so that its
fluorescence increases drastically. This can be also understood from
the fact that compounds with acyclic C]N double bonds are
N
N
Z
N
O
O
N
O
O
N
O
O
CH CN
Zn
X
IC
IC / Zn
Minor control unit to tune selectivity
(b
)
H
N
H
Zn2+
N
N
N
N
N
O
O
Zn
N
Aqueous Solution
N
O
O
Cl
IC1 / Zn2+
Cl
IC1
Fig. 1. Proposed sensing mechanism based on (a) C]N isomerisation between IC and
Zn2þ, and (b) the controllable C]N isomerisation between IC1 and Zn2þ
* Corresponding author. E-mail address: wangpf@mail.ipc.ac.cn (P. Wang).
.
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2012.04.090

J. Wu et al. / Tetrahedron 68 (2012) 5458e5463
5459
hydrazine could form a pseudocavity with different affinity abilities
(binding constants) to Zn2þ and Cd2þ, which is possible to serve as
a turn-on sensor to discrimination Zn2þ from Cd2þ ions. Compared
with previous sensor IC, the modified sensor IC1 shows some ad-
vantages, i.e., methyl is used to tune the conformational change;
pyridyl hydrazine in IC1 shows good water solubility and its strong
affinity to Zn2þ than antipyrine in IC. As expected, IC1 and IC2 give
rise to significant fluorescence enhancements to Zn2þ in aqueous
solution due to the suppression of C]N isomerization. Moreover,
IC1 displays much higher selectivity to Zn2þ over Cd2þ compared
with IC2, indicating that the introduction of methyl to imine does
play an important role to tune its sensing selectivity for C]N
isomerization-based sensors by a fine tune mechanism. In addition,
IC1 was further utilized to image intracellular Zn2þ ions in HepG2
cells.
a new absorption band at 466 nm emerged with increasing in-
tensity, whereas that at 418 nm disappeared completely. Mean-
while, a distinct isosbestic point at 432 nm was also observed,
implying the formation of a 1:1 complex. Job’s plot also indicated
that IC1 can bind Zn2þ ions with 1:1 stoichiometry (Fig. S1y). Cor-
respondingly, fluorescence titration spectra of IC1 with Zn2þ were
given in Fig. 2b. It was found that the fluorescence of IC1 in CH3CN/
H2O buffer solution increases dramatically with increasing con-
centrations of Zn2þ, concomitantly the maximum emissive wave-
length shifts from 523 nm to 509 nm. Actually, an obviously green
emission of the solution can be easily observed by the naked eyes.
The binding constant between IC1 and Zn2þ in CH3CN/H2O buffer
solution was calculated to be 3.3ꢀ106 Mꢁ1 from the fluorescence
titration data in Fig. 2b.9 Fluorescence quantum yield (Ff) of free IC1
is 0.014, whereas it reaches 0.26 when IC1 binds with Zn2þ (fluo-
rescein in 0.1 N NaOH as a reference, Ff¼0.85).10 Obviously, the free
IC1 is non-fluorescent because the C]N isomerization is the pre-
dominant decay process in the excited states. In contrast, its com-
plexation with Zn2þ ions restricts the rotation of C]N bonds and
results in the suppression of C]N isomerization so that its fluo-
rescence is drastically increased.
2. Results and discussion
In our present work,
a
typically fluorescent dye 7-
diethylaminocoumarin was selected not only as a fluorophore but
also as a receptor providing a binding site. As described in Scheme
1, a ketone (1) or aldehyde (2) in the 3-position of coumarin fluo-
rophore was obtained from diethylaminosalicylaldehyde by two
different condensation reactions. 2-Hydrazinopyridine, as an ad-
ditional chelating moiety, was connected with 1 or 2 via C]N bond
to form two potential fluorescence turn-on sensors (IC1 and IC2,
respectively) for Zn2þ ions. IC1 and IC2 were characterized by 1H
NMR, 13C NMR, HRMS and the synthetic details were given in the
experimental section.
The explicit structure of complex IC1/Zn2þ crystallized from
THF/diethyl ether is determined from X-ray diffraction analysis. As
shown in Fig. 3, IC1 and ZnCl2 form a complex with 1:1 stoichi-
ometry. Zn2þ locates at the centre of the pseudocavity of IC1 and
coordinates with oxygen atom of carbonyl group, two N atoms of
imine and pyridine, and two Cl atoms from the counter anions to
form a distorted tetrahedron structure. Such a crystal structure
restricts the free rotation of C]N bonds of IC1 and its fluorescence
is thus turn-on.
To further get insight into the C]N isomerization-based decay
process, the fluorescence lifetime (s) of IC1 in CH3CN/H2O was
measured by single photon counting and it shows a good single-
H
CHO
OH
N
a
O
b
N
N
O
N
O
N
O
N
O
1
IC1
c, d
H
CHO
O
N
N
e
b
N
O
N
O
O
N
O
N
O
IC2
2
Scheme 1. Synthetic pathway of IC1 and IC2: (a) Ethyl acetonacetate/Piperidine/EtOH,
reflux 5 h; (b) 2-Hydrazinopyridine, EtOH, rt 15 h; (c) Diethyl malonate/EtOH, reflux
6 h; (d) HCl/HOAc, reflux 10 h; (e) POCl3/DMF, 60 ꢂC, 10 h.
Fig. 2a shows the UVevis absorption spectra of IC1 in the
presence of different concentrations of Zn2þ ions. The free IC1 has
a characteristic absorption band peaked at 418 nm in CH3CN/H2O
buffer solution. Upon gradual addition of Zn2þ to the solution of IC1,
Fig. 3. ORTEP diagram of the complex IC1/ZnCl2 with displacement atomic ellipsoids
drawn at 30% probability level.
0.5
(a)
(b)
1000
2+
0.4
Zn
2+
Zn
800
600
400
200
0
0.3
0.2
0.1
0.0
300
400
500
600
450
500
550
600
Wavelength (nm)
Wavelength (nm)
Fig. 2. Absorption spectra (a) and fluorescence spectra (b) of IC1 (10
m
M) in CH3CN/H2O solution (v/v¼3/7, TriseHCl 10 mM, PH¼7.4) upon addition of different concentrations of
Zn(ClO4)2 (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 M) with an excitation wavelength of 432 nm.
m

5460
J. Wu et al. / Tetrahedron 68 (2012) 5458e5463
exponential decay (Table 1). The lifetime of free IC1 is quite short
(<0.10 ns). The radiative decay rate constant (kr) and nonradiative
decay rate constant (knr) are calculated to be about 1.4ꢀ108 sꢁ1 and
results indicate that C]N isomerization as the nonradiative decay
is not a predominant process in the excited state of IC2. It is con-
cluded that the methyl in IC1 can be as a minor unit to control C]N
isomerization. The introduction of methyl group to IC1 maybe
change the nonradiative decay (isomerization, polarization and
planarity) of imine moiety, which may further improve its sensing
selectivity to Zn2þ accordingly.
9.9ꢀ109
s
ꢁ1, indicating that the nonradiative decay is the pre-
dominant process in the excited states of IC1.11 When Zn2þ was
added, the lifetime of IC1 increases to 0.86 ns, which was much
longer than that of free IC1, and the radiative and nonradiative
decay rate constants were changed to 3.0ꢀ108 sꢁ1 and 8.6ꢀ108 sꢁ1
,
Variation of fluorescence spectra of IC1 and IC2 upon addition of
respectively; both radiative and nonradiative decay processes be-
different metal ions including Cd2þ, Pb2þ, Zn2þ, Ca2þ, Naþ, Mg2þ
,
came comparative so that the fluorescence was increased.
Hg2þ, Fe2þ, Co3þ, Ni2þ, Cu2þ ions, were shown in Fig. 4. For IC1, Zn2þ
gave rise to the largest enhancement (18.5-fold in fluorescence
quantum yield) among these metal ions; Cd2þ induced only a few
fluorescence increases, whereas Cu2þ, Co3þ and Ni2þ induced dis-
tinct fluorescence quenching of IC1 (Fig. 4a). Other ions including
Pb2þ, Hg2þ, Fe2þ, Ca2þ, and Naþ did not cause any obvious changes
in fluorescence, indicating that IC1 shows a selective fluorescence
increase to Zn2þ. However, for IC2, Zn2þ only induced a 2.2-fold
increase in fluorescence quantum yield, whereas Cd2þ also resul-
ted in a 1.7-fold increase. In addition, Cu2þ, Co3þ and Ni2þ caused
distinct fluorescence quenching of IC2 (Fig. 4b). Compared with
Fig. 4a and b, IC1 shows a better selective fluorescence increase to
Zn2þ than IC2. Different fluorescence changes of IC1 and IC2 with
various cations can be understood as follows. Generally, transition
ions with open shell d-orbitals often quench the fluorescence of
fluorophores due to the electron/energy transfer between the
metal cations and fluorophores, providing a very fast and efficient
nonradiative decay process in the excited states.7b,14 In contrast, the
transition ions with close shell d-orbitals, such as Zn2þ, do not in-
troduce low energy metal-centred or charge-separated excited
states, so the energy/electron transfer processes cannot take place.
In the case of IC1 and IC2, we observed an obvious fluorescence
enhancement in the presence of Zn2þ, and fluorescence quenching
in the presence of Cu2þ, Co3þ and Ni2þ. The reason can be explained
by a competition process between the fluorescence enhancement
due to the inhibition of C]N isomerization and quenching of
fluorescence from the metal ion-induced electron/energy transfer
processes.7b
Table 1
Photophysical data of IC1 and IC2 in the absence and presence of Zn2þ ions
Sensors
labs
lem
ε
Vf
s (ns)
(nm)
(nm)
(Mꢁ1 cmꢁ1
)
(quantum yield)
IC1
418
466
452
472
519
509
530
513
3.3ꢀ104
3.9ꢀ104
4.5ꢀ104
5.7ꢀ104
0.014
0.26
0.20
0.44
<0.10
0.86
0.63
1.31
IC1þZn2þ
IC2
IC2þZn2þ
To illustrate the important role of methyl group in IC1 to tune its
sensing selectivity to Zn2þ, a methyl-free IC2 as a reference sensor
was synthesized and its sensing activity was also studied. As shown
in Fig. S2y, IC2 showed similar UVevis and fluorescence responses
to Zn2þ as IC1 did. For example, upon complexation with Zn2þ in
aqueous solution, the characteristic absorption wavelength of IC2
shifted from 452 nm to 472 nm; its fluorescence spectrum also
displayed a distinct enhancement. However, IC2 only induced
a smaller increase in its fluorescence quantum yield from 0.20 to
0.44, which is much lower than that of IC1. Job’s plot and MS
analyses implied that IC2 and Zn2þ form a complex with 2:1 stoi-
chiometry, which is different from that of IC1/Zn2þ (Fig. S1y and
S4y). We ascribed the fluorescence increase of IC2 with Zn2þ
mainly to the suppression of C¼N isomerization. The distinct dif-
ference of fluorescence quantum yields of free IC1 and IC2 can be
understood not only from C]N isomerization but also from other
non-radidative decay. The direct introduction of methyl to IC1
maybe increase the molecular polarization and planarity, which
increases its fluorescence accordingly.12 In addition, the intra-
molecular quenching between the two fluorophores that will de-
crease the fluorescence of Zn(IC2)2 to some extent is another
The fluorescence response of IC1 to Zn2þ in the presence of
various competing cations and its selectivity for Zn2þ are illustrated
in Fig. 5. The Zn2þ response of IC1 is unaffected in a background of
toxic heavy metal ions, such as Pb2þ, Cd2þ and Hg2þ. It is very
important that IC1 can distinguish Zn2þ from these toxic heavy
metal ions, especially from Cd2. All tested cations, in addition to
Cu2þ, Co3þ and Ni2þ, do not inhibit the fluorescence increase of IC1
to Zn2þ. However, IC2 does not exhibit a selective sensing to Zn2þ
over Cd2þ, indicating that the methyl group in IC1 does play an
possible reason. The lifetime of free IC2 (
s
¼0.63 ns) is much longer
than that of IC1 (<0.10 ns). Different photophysical data of IC1 and
IC2 with Zn2þ are consistent with their different binding modes.
The possible reason for the formation of different stiochiometry is
that the introduction of methyl onto the imine may distinctly affect
the steric hindrance and electronic structure of the imine.13 These
important role to tune its sensing selectivity to Zn2þ over Cd2þ
.
High selectivity to Zn2þ can be explained via different binding
constants. Suppose IC1 and IC2 interact with various cations with
1200
6000
2+
Zn
(a)
(b)
2+
Zn
1000
5000
800
600
4000
2+
Cd
2+ 2+
IC1, Pb , Hg , Ca
2+
2+ 2+
IC2, Hg , Ca
3000
2000
1000
0
2+
Pb
2+
2+
Fe
400
200
0
2+
Fe
2+
Cd
Cu
2+ 3+
Cu , Co , Ni
2+
2+
Ni
3+
Co
500
550
Wavelength (nm)
600
450
500
550
Wavelength (nm)
600
Fig. 4. Fluorescence spectra of (a) IC1 (10
(each 30
m
M) and (b) IC2 (10 m
M) upon addition of different metal ions including Cd2þ, Pb2þ, Zn2þ, Hg2þ, Cu2þ, Fe2þ, Co3þ, Ni2þ, Ca2þ, Mg2þ, Naþ
m
M) in CH3CN/H2O (3/7, TriseHCl 10 mM, PH¼7.4) buffer solution.

J. Wu et al. / Tetrahedron 68 (2012) 5458e5463
5461
3. Conclusions
8
6
4
2
0
In summary, we have developed a new turn-on fluorescent
sensor (IC1) for Zn2þ both in aqueous solution and in living cells.
IC1 can well distinguish Zn2þ from Cd2þ by the fine control of C]N
isomerization, which is difficult to accomplish from other con-
ventional designs. We believe that such a controllable conforma-
tional change can be used as a general principle to extend to other
sensing systems for improvement of the selectivity, especially for
specific recognition of Zn2þ from Cd2þ in aqueous solution. The
present work provides a new concept to control the excited state of
C]N isomerization via a minor control unit.
4. Experimental
2+
2+
2+
2+
2+
2+
2+
+
Na Co
3+
2+
Ni Cu
2+
Zn
Cd
Pb
Hg
Fe
Ca
Mg
4.1. Method and measurement
Fig. 5. Fluorescence responses of IC1 (10
presence (red bar) of various competing ions including Cd2þ, Pb2þ, Hg2þ, Fe2þ, Ca2þ
Naþ, Mg2þ, Co3þ, Ni2þ, Cu2þ (each 30
M) in CH3CN/H2O (3/7, TriseHCl 10 mM,
m
M) to Zn2þ in the absence (black bar) and
1H NMR and 13C NMR spectra were recorded on a Varian Gemin-
300 (400 MHz). UVevis absorption and fluorescence spectra were
recorded in Hitachi U-3010 spectrometer and Hitachi F-4500
fluorescence spectrometer, respectively. Mass spectra were recor-
ded on a Finigan 4021C MS-spectrometer. X-ray analysis was
measured on Rigaku R-Axis Rapid IP diffractometer. Fluorescence
images in living cells were recorded in a Nikon C1si laser scanning
confocal microscope and multiphoton system (confocal excitation:
an Ar ion laser at 488 nm).
4-Diethylaminosalicylaldehyde, 2-hydrazinopyridine, diethyl
malonate, ethyl acetonacetate, and cationic compounds such as
Cu(ClO4)2, Zn(ClO4)2, Pb(ClO4)2, Cd(ClO4)2, Fe(ClO4)2, Hg(ClO4)2,
Ca(ClO4)2, NaClO4, were purchased from Aldrich and used as re-
ceived. CoCl3 and NiCl2 were from Beijing Chemical works and used
as received. All other reagents are analytical grade and purchased
from Beijing Chemical works.
,
m
PH¼7.4) buffer solution (excitation at 432 nm). Bars represent the final integrated
fluorescence response (Ff) over the initial integrated emission (Fi).
1:1 and 2:1 based on their binding ratio with Zn2þ, their binding
constants can be estimated as shown in Table 2.9 Because the
binding constant of IC1/Zn2þ is two orders of magnitude higher
than that of IC1/Cd2þ, IC1 displays a good selectivity to Zn2þ over
Cd2þ. The binding constants of IC1/Zn2þ and IC1/Cu2þ are com-
parative, thus IC1 cannot recognize Zn2þ over Cu2þ. Likewise, IC2
does not display a good selectivity to Zn2þ over Cd2þ because their
binding constants are comparative.
For the practical application of IC1, its fluorescence imaging for
Zn2þ was examined in living cells using confocal fluorescence im-
A 1 mM of IC1 (or IC2) was prepared in buffer solution CH3CN/
H2O (v/v¼3/7, 10 mM, TriseHCl, pH¼7.4) as the stock solution.
Cationic stock solution were prepared in buffer solution CH3CN/
H2O (v/v¼3/7, 10 mM, TriseHCl, pH¼7.4) and their concentrations
are all 1 mM for fluorescence and UVevis spectra analysis. These
solutions were kept at about 4 ꢂC for further use. Excitation
wavelength of IC1 was 432 nm and the temperature is 25 ꢂC.
Spectra analysis of model compound IC2 is the same as that of IC1,
but the excitation wavelength changes to 450 nm.
aging (Fig. 6). Incubation of HepG2 cells with IC1 (20 mM) for 10 min
then washing carefully, almost no fluorescence could be observed
(Fig. 6b). These living cells were then incubated with Zn2þ
(0.1 mM), and a green fluorescence emission was observed after
6 min incubation (Fig. 6c). The emission became very strong after
15 min (Fig. 6d), illustrating that IC1 can be employed successfully
as a fluorescence probe for Zn2þ imaging in HepG2 cells.
Fluorescence quantum yields were determined by comparing
the emission integral area of the sample with that of the fluores-
cence reference based on the following equation:
Table 2
Binding constants of IC1 and IC2 with Zn2þ, Cd2þ, Hg2þ, Pb2þ, Cu2þ ions
Sensors
Binding
Sensors
Binding
!
ꢀ
ꢁ
constant (Mꢁ1
)
constant (Mꢁ2
)
2
2
AU
AR
nU
nR
FU
¼
FR
IC1þZn2þ
IC1þCd2þ
IC1þHg2þ
IC1þPb2þ
IC1þCu2þ
3.3ꢀ106
5.5ꢀ104
N.A.a
IC2þZn2þ
IC2þCd2þ
IC2þHg2þ
IC2þPb2þ
IC2þCu2þ
1.2ꢀ108
7.5ꢀ107
N.A.
where, AU and AR are the integrated area under the corrected
fluorescence spectra for the sample and reference, nU and nR are the
refractive indexes of the sample and reference, respectively. Fluo-
rescence quantum yield used for the reference is the fluorescein in
N.A.
N.A.
1.8ꢀ106
2.8ꢀ108
a
Too low to be calculated.
Fig. 6. Confocal images of HepG2 cells. Bright-field image (a); HepG2 cells incubated with IC1 (20
HepG2 cells in b further incubated with Zn(ClO4)2 (0.1 mM) for 6 min (c) and 15 min (d).
mM) in CH3CN/H2O buffer (v/v¼3/7, TriseHCl, 10 mM, PH¼7.4) for 10 min (b);

5462
J. Wu et al. / Tetrahedron 68 (2012) 5458e5463
0.1 N NaOH aqueous solution, which has a known quantum yield of
0.85.10b
ArH), 6.62e6.59 (m, 1H, ArH), 6.50 (s, 1H, ArH), 3.46e3.40 (m, 4H,
CH2),1.24e1.21 (t, 6H, CH3).13C NMR (100 MHz, CDCl3)
162.0,156.5,
d
The radiative decay rate constant (kr) and nonradiative decay
rate constant (knr) are calculated by the equation:
156.4, 151.0, 147.8, 138.3, 136.9, 134.1, 129.8, 116.0, 114.7, 109.5, 109.1,
107.5, 97.5, 45.1, 12.6. TOF MS-EI: 336.1330 (calculated: 336.1586).
ꢂ
ꢃ.
Kr
¼
Ff
=
s
; and knr
¼
1 ꢁ Ff
s
4.3. Crystal structure
where Ff is the fluorescence quantum yield and
s is the fluores-
Crystal data and details for the data collection of the complex
IC1/ZnCl2 were provided in Table 3. The diffraction data were col-
lected on a Bruker SMART D8 goniometer with APEX CCD detector,
using MoKa radiation l¼0.71073 P (graphite monochromator). The
structure was solved by a direct method (SHELXTL) and refined on
F2 by full-matrix least-squares techniques.15 Hydrogen atoms were
included using a riding model. CCDC-852468 for the complex IC1/
ZnCl2 contains the supplementary crystallographic data for this
paper. These data can be obtained free of charge from the Cam-
bridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/
data_request/cif.
cence lifetime.
4.2. Synthesis
4.2.1. 7-N,N-Diethylaminocoumarin-3-acetone
(1). 4-N,N-Dieth-
ylaminosalicyaldehyde (19.3 g, 0.1 mol), ethyl acetonacetate (18.5 g,
0.15 mol), and 1 mL of piperidine were dissolved in 200 mL of ab-
solute ethanol. After the mixture solution was refluxed for 5 h, the
solvent was removed under reduced pressure. The yellow solid was
precipitated and collected, and the product was recrystallized from
absolute ethanol to afford 1 (yield: 82%). 1H NMR (400 MHz, CDCl3)
d
8.42e8.41 (d, 1H, ArH, J¼2.2 Hz), 7.39e7.37 (m, 1H, ArH),
Table 3
Crystal data and structure refinement for IC1/ZnCl2
6.62e6.59 (m, 1H, ArH), 6.46e6.45 (d,1H, ArH, J¼2.4 Hz), 3.47e3.42
(q, 4H, CH2), 2.67 (t, 3H, COCH3), 1.25e1.21 (t, 6H, CH3). 13C NMR
Empirical formula
C20H22Cl2N4O2Zn
20H22Cl2N4O2Zn
486.69
(100 MHz, CDCl3)
110.0, 108.3, 96.7, 45.3, 30.7, 12.5. TOF MS-EI: 259.1230 (calculated:
259.1208).
d 195.8, 161.0, 158.9, 153.1, 148.0, 132.0, 116.2,
Empirical formula
Formula weight
Temperature
C
173(2) K
ꢀ
Wavelength
0.71073 A
Crystal system, space group
Unit cell dimensions
Monoclinic, P2(1)/c
4.2.2. (E)-7-(diethylamino)-3-(1-(2-(pyridin-2-yl)hydrazono)ethyl)-
2H-chromen-2-one (IC1). To the intermediate 7-N,N-dieth-
ylaminocoumarin-3-acetone (259 mg, 1 mmol) dissolved in 20 mL
of ethanol, was added 2-hydrazinopyridine (120 mg, 1.1 mmol)
dissolved in 5 mL of ethanol dropwise. After the reaction solution
was stirred at room temperature for 15 h, the solvent was removed
under reduced pressure. The residue was purified by flash chro-
matography (eluent EtAc/Hexane¼1/3) to give IC1 as an orange
ꢀ
a¼10.021(2) A
a
¼90 deg
ꢀ
B¼15.138(3) A
b
¼102.78(3) deg
ꢀ
c¼14.044(3) A
g¼90 deg
3
ꢀ
Volume
2077.7(7) A
Z, Calculated density
Absorption coefficient
F(000)
Crystal size
Theta range for data collection
Limiting indices
Reflections collected/unique
Completeness to theta¼25.00
Absorption correction
Max. and min. transmission
Refinement method
Data/restraints/parameters
Goodness-of-fit on F^2
Final R indices [I>2sigma(I)]
R indices (all data)
4, 1.556 mg/m3
1.464 mmꢁ1
1000
0.21ꢀ0.20ꢀ0.12 mm
2.08e25.00 deg.
needle crystal. (yield: 35.0%) 1H NMR (400 MHz, CDCl3)
d 8.15e8.13
ꢁ11<¼h<¼11,ꢁ18<¼k<¼18,ꢁ16<¼l<¼16
6754/3642 [R(int)¼0.0339]
99.8%
(d, 1H, ArH, J¼4.8 Hz), 8.10 (s, 1H, ArH), 7.92 (s, 1H, NH), 7.61e7.57
(m, 1H, ArH), 7.34e7.32 (t, 2H, ArH), 6.79e6.75 (q, 1H, ArH),
6.60e6.57 (d, 1H, ArH, J¼8.8 Hz), 6.49e6.48 (s, 1H, ArH), 3.44e3.35
(m, 4H, CH2), 2.29 (s, 3H, COCH3), 1.23 (t, 6H, CH3). 13C NMR
Semi-empirical from equivalents
0.8439 and 0.7486
Full-matrix least-squares on F2
3642/0/262
(100 MHz, CDCl3)
d 161.6, 157.0, 156.8, 150.9, 147.9, 142.7, 141.1,
1.236
138.2, 129.7, 119.9, 116.1, 109.2, 108.9, 107.6, 97.1, 45.0, 14.5, 12.6. TOF
MS-EI: 350.1346 (calculated: 350.1743).
R1¼0.0625, wR2¼0.1121
R1¼0.0815, wR2¼0.1179
ꢀꢁ3
Largest diff. peak and hole
0.549 and ꢁ0.502 e A
4.2.3. 7-N,N-Diethylaminocoumarin-3-aldehyde
(2). The
in-
termediate 2 was synthesized according to the reference with
a minor modification.7b To a 50 mL two-neck flask, were added
2.5 mL of POCl3 and 3.5 mL of DMF under N2. After the solution was
heated at 60 ꢂC for 2 h, a suspended DMF solution of 7-N,N-
diethylaminocoumarin (2.0 g, 10 mL) was added dropwise and the
reaction was heated at 60 ꢂC for another 10 h. When the formed
dark brown solution was poured into 100 mL of ice-water, a dark-
orange solid was precipitated out. The crude product was recrys-
tallized from absolute ethanol to afford an orange needle crystal
4.4. Cell culture
HepG2 cells were prepared from continuous culture in MEM
mixed with 10% foetal bovine serum (FBS), 1% penicillin/strepto-
mycin at 37 ꢂC in 5% CO2 humidified air. Cell culture medium was
removed after the cell adhesion for 24 h. Following the digestion
with trypsin, 1.0 mL of cell suspension was incubated in cell walls of
the micro plate at 37 ꢂC in 5% CO2 humidified air for 24 h. The cell
layer was washed twice with phosphate buffered saline (PBS), and
1.0 mL of PBS was added into each well for fluorescence imaging.
HepG2 cells were incubated with IC1 for several minutes at room
temperature. After the cell layer was carefully washed twice with
phosphate buffered saline (PBS), the fluorescence imaging was
imaged using a Nikon confocal microscope. Excitation wavelength
of laser was 408 nm and the emission was recorded at 515 nm. EZ-
C1 3.20 free viewer was used as a platform for data analysis.
(yield: 70%). Mp 168e170 ꢂC. 1H NMR (400 MHz, CDCl3)
d 10.13 (s,
1H, CHO), 8.26 (s, 1H, ArH), 7.43(d, 1H, J¼8.9 Hz, ArH), 6.67 (d, 1H,
J¼7.6 Hz, ArH), 6.50(S, 1H, ArH), 3.49 (m, 4H, CH2), 1.25 (t, 6H, CH3).
4.2.4. (E)-7-(Diethylamino)-3-((2-(pyridin-2-yl)hydrazono)methyl)-
2H-chromen-2-one (IC2). To 20 mL of the intermediate 7-N,N-
diethylaminocoumarin-3-aldehyde (250 mg, 1 mmol) in ethanol,
was added 5 mL of 2-hydrazinopyridine (120 mg, 1.1 mmol) in
ethanol dropwise. After the reaction solution was refluxed for 3 h,
the orange solid was precipitated, collected, and washed with eth-
Acknowledgements
anol to afford IC2 (yield: 71%). 1H NMR (400 MHz, CDCl3)
d 8.64 (s,
1H, CH]N), 8.19 (s, 1H, ArH), 8.16e8.14 (t, 1H, ArH), 7.96 (s, 1H, NH),
7.63e7.58 (m, 1H, ArH), 7.36e7.32 (t, 2H, ArH), 6.79e6.76 (m, 1H,
This work was supported by the NNSF of China (Grant Nos.
21173244 and 21073213), the Main Direction Program of

J. Wu et al. / Tetrahedron 68 (2012) 5458e5463
5463
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Supplementary data
NMR and MS copies of new compounds, additional spectro-
scopic data and the cif file of the complex IC1/ZnCl2. Supplemen-
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