A selective NIR-emitting zinc sensor by using Schiff base binding to turn-on excited-state intramolecular proton transfer

Article abstract of DOI:10.1039/c3tb21339k

A rational design has led to a highly selective and cell-permeable zinc sensor, which exhibits not only a large fluorescence turn-on at ～545 nm but also the desirable NIR emission (～720 nm) with a large Stokes' shift, providing a practical sensor platform

Full text of DOI:10.1039/c3tb21339k

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Materials Chemistry B
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A selective NIR-emitting zinc sensor by using
Schiff base binding to turn-on excited-state
intramolecular proton transfer†
Cite this: J. Mater. Chem. B, 2014, 2,
2008
Junfeng Wang,a Yingbo Li,c Ernest Duah,a Sailaja Paruchuri,a Demin Zhouc
ab
*
and Yi Pang
Received 26th September 2013
Accepted 23rd January 2014
A rational design has led to a highly selective and cell-permeable zinc sensor, which exhibits not only a large
fluorescence turn-on at ꢀ545 nm but also the desirable NIR emission (ꢀ720 nm) with a large Stokes' shift,
providing a practical sensor platform with two emission channels for reliable zinc detection.
DOI: 10.1039/c3tb21339k
www.rsc.org/MaterialsB
Signicant interest exists to detect and quantify the dynamic the phenolic proton in HBO, thereby disabling the ESIPT
zinc ion ux in live tissues or animals, as zinc is an essential mechanism and diminishing the Stokes' shi.
element which plays important roles in many cellular processes
Recently, our group has designed the bis(HBO) 3-Zn, by
including gene expression and signal transduction.1 For in vivo changing the R1 and R2 substituents in 1-Zn to respective ben-
applications,2 the molecular probe is required to emit optical zoxazole and hydroxy groups to introduce the 2nd HBO.10–12
signals in the near infrared (NIR) region (700–900 nm), as NIR Upon binding to Zn2+ cations, the weak uorescence of 3 is
light can penetrate more deeply into biological tissues.3 turned on, giving both green (lem z 540 nm) and NIR emission
Although many zinc probes are available,4 few can give NIR (lem z 750 nm). The NIR turn-on signal from 3, however, also
emission,5,6 and most are based on cyanine dyes. The cyanine responds to Cd2+ cations.11 It remains a challenge to develop a
dyes, however, exhibit a small Stokes' shi (typically about 20– Zn2+ sensor that gives desirable NIR emission without inter-
50 nm), which hampers their broad applications. For example, a ference from the structurally similar Cd2+ cations. In addition,
cyanine-based zinc sensor has a 730 nm excitation wavelength the intensity of the desirable NIR emission from 3-Zn also needs
and a 780 nm emission wavelength.5 It remains a challenge to to be further tuned. And the synthesis of the sensor needs to be
develop a Zn2+ sensor that exhibits not only desirable NIR simplied for practical applications.
emission, but also large Stokes' shi and high selectivity (e.g.
In an effort to tune the performance of the ESIPT probe, the
without interference from the structurally similar Cd2+ cations). complex 4-Zn appears to be an interesting system. In compar-
2-(20-Hydroxyphenyl)benzoxazole (HBO) 1 has emerged to be ison with 3-Zn, the complex 4-Zn uses an isolated imine bond
an interesting component in the sensor design,7,8 as it can (ꢁCH]Nꢁ) to bind Zn2+ cations. When the zinc-binding ben-
undergo the excited-state intramolecular proton transfer zoxazole fragment in 3 is replaced by a –CH]Nꢁ bond, one of
(ESIPT) to produce the emission with a large Stokes' shi (ca. the two HBO units in bi(HBO) 3 is removed, which could
150–200 nm). Among the few HBO derivatives for zinc detec- inuence the ESIPT signal of the neutral ligand and its metal
tion, O'Halloran's group reported 1a for intracellular Zn2+ complexes. In addition, the zinc-chelation of 4 will form a more
sensing, via formation of zinc complex 1a-Zn (lmax ¼ 376 nm, stable ve-membered ring “A” involving the –CH]Nꢁ and
lem ¼ 443 nm).7 Kwon and co-workers reported another HBO pyridyl groups, in contrast to a six-membered ring in 3-Zn that
derivative 1b (lmax ¼ 379 nm, lem ¼ 550 nm), whose zinc involves the benzoxazole and pyridyl groups. Such structural
complex 1b-Zn gives enhanced emission (lmax ¼ 443 nm, lem
¼
change could perturb the interaction between the zinc cation
542 nm).9 Formation of zinc complex 1-Zn, however, removes and phenolic oxygen, thereby tuning the ESIPT of the 2nd HBO
unit.
While some Schiff-base chemosensors are known for selec-
tive Zn2+ detection,13–17 they give uorescence either in the
aDepartment of Chemistry, The University of Akron, Akron, Ohio 44325, USA. E-mail:
blue14,16,17 or green colors13,15 with small Stokes' shi (ꢀ30 nm).
yp5@Uakron.edu
bMaurice Morton Institute of Polymer Science, The University of Akron, Akron, Ohio
In addition, the known Schiff-base sensors are dependent on
the uorescence turn-on through metal binding-induced
isomerization13 of the –C]Nꢁ bond with little spectral shi.
Reasoning that using a Schiff base ligand could improve the
selectivity in Zn2+ binding, we decided to explore the mono HBO
44325, USA
cState Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical
Sciences, Peking University, Beijing 100191, China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c3tb21339k
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sensor 4. Intriguingly, the sensor 4 exhibited great selectivity
toward Zn2+ binding, which turns on the ESIPT to give the
desirable NIR emission (at ꢀ720 nm) with a large Stokes' shi
(ꢀ260 nm). The ndings demonstrate that a Schiff base can
serve as an effective switch for the ESIPT turn-on, whose excel-
lent selectivity to bind zinc cations makes the NIR sensor an
attractive candidate for practical applications.
Synthesis of 4 was accomplished by reaction of 2-hydro-
zinylpyridine with the corresponding aldehyde 6 in high yield
(Scheme 2). The simplicity in the synthesis of the ligand 4 was in
sharp contrast to that of ligand 3 which used a sequence of four
synthetic steps from the aldehyde (with low yield).12 The
binding properties of 4 to Zn2+ cations were examined in EtOH/
HEPES (Fig. 1). As the Zn2+ cations were added, a new absorp-
tion band was generated at about 450 nm. The new band was
attributed to the removal of a phenolic proton as shown in the 4-
Zn complex. The signal change appeared to be complete when
one equivalent Zn2+ was added, suggesting a 1 : 1 ligand-to-
Scheme 2 Synthesis of sensor 4.
metal ratio (ESI Fig. S3†). Addition of other cations, such as Al3+
,
however, did not cause a notable bathochromic shi, indicating
that their bindings were not sufficiently strong to remove the
phenolic proton (Fig. 1, S5 and S6†). The sensor also showed
weak binding to Cd2+ cations (Fig. 1).
The ligand 4 gave a weak uoresence peak at 580 nm (f ¼
0.05). Upon addition of Zn2+ cations, the uorescence signi-
cantly increased (f ¼ 0.26 in 1 : 1 EtOH/H2O), giving two new
emission peaks at 545 and ꢀ720 nm (Fig. 2a and S2†, and Table
1), which correspond to the emission from the enol and keto
tautomers of 4-Zn, respectively. NIR emission was relatively
Fig. 1 Absorbance spectra of 4 (10 mM) in EtOH/HEPES (10 mM) (1 : 1)
(pH ¼ 7.2), upon addition of 5 equiv. of different cations.
Table 1 Absorption and emission of ESIPT sensors
Absorption
Emission
lmax (nm)
lem (nm)
Stokes' shi
1a
1a-Zn
1b
1b-Zn
3
3-Zn
4
337
376
379
443
412
465
413
450
407
443
550
542
5103 cmꢁ1 (70 nm)
4022 cmꢁ1 (67 nm)
8203 cmꢁ1 (171 nm)
4123 cmꢁ1 (99 nm)
7878 cmꢁ1 (198 nm)
8172 cmꢁ1 (285 nm)
6971 cmꢁ1 (167 nm)
8333 cmꢁ1 (270 nm)
456, 610a,11
546, 750a
580
4-Zn
545, 720a
a
Indicates the peak used to calculate Stokes' shi.
stronger in pure methanol (ESI Fig. S4†). The green uorescence
turn-on could be easily observed, making it also useful for
naked eye detection (Fig. 2b). Interestingly, addition of other
cations (K+, Na+, Ag+, Cd2+, Ca2+, Mg2+, Pb2+, Ni2+, Hg2+, Co2+,
Cu2+, Cr3+, Fe3+, Al3+) to 4 did not induce the dual emissions at
545 and 720 nm. In other words, the probe molecule 4 is silent
to Cd2+ cations, as its binding could not remove the phenolic
proton (a necessary step to simultaneously shi the emission
and turn on the ESIPT for NIR emission). The Al3+ cations only
induced an enhanced emission at ꢀ610 nm, as its stronger
Lewis acid character could lead to a moderately strong inter-
action with the phenol segment (Fig. S6†). In summary, the
sensor 4 displayed an excellent selectivity toward Zn2+ binding,
Scheme 1 Structures of 2-(20-hydroxyphenyl) benzoxazole (HBO)
derivatives, where the original HBO unit is shown in blue.
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2-(hydrozino)pyridine20 unit in 4a-Zn can be tautomerized to
give 4b-Zn (Scheme 1), which might also contribute to the signal
broadening from the 1H NMR. Mass spectrometry detected m/z
¼ 422.0496 by HRMS (Fig. 4), corresponding to [4b-Zn ꢁ L]
(calcd for C20H14N4O3Zn: 422.0357, see ESI Fig. S14†).
In order to clarify the interactions of 4 with different metal
ions, the UV-vis absorption and uorescence spectra of 4 (10 mM
in EtOH : H2O ¼ 1 : 1 solutions) were studied by addition of 5
equiv. of different cations (ESI Fig. S5 and S6†). In addition to
no response to physiologically important K+, Na+, Ca2+ ions, the
sensor also exhibited no observable uorescence response to
Cd2+ cations, which led to greatly improved selectivity over the
previous sensor 3.9,10 While some cations (Ag+, Co2+, Ni2+ and
Cu2+) quenched the uorescence, addition of Cr3+, Fe3+ and Al3+
cations slightly increased the uorescence whose signals were
red-shied by ꢀ25–37 nm. Only Zn2+ binding gave enhanced
emissions at 545 nm and ꢀ720 nm, which are signicantly
shied from the neutral ligand (580 nm) to allow the positive
identication of the zinc cations.
Fig. 2 (a) Fluorescence spectra of 4 (10 mM) in EtOH : HEPES ¼ 1 : 1
(pH ¼ 7.2) upon addition of 5 equiv. of different ions. (b) Fluorescence
As revealed from Fig. 2, only zinc binding to 4 could effec-
tively turn on the ESIPT, giving enhanced emission from both
enol (lem z 545 nm) and keto tautomers (lem z 720 nm). A
peculiar question is why the ligand 4 can bind Zn2+ cations well,
while being silent to the Cd2+ cations with similar atomic
conguration ([Ar]3d10 for Zn2+ and [Kr]4d10 for Cd2+).21 On the
basis of quick disappearance of the –OH signal at ꢀ10 ppm (1H
NMR in Fig. 3), the initial step might involve a weak interaction
of a metal cation with the imine –CH]N-, forming intermediate
5. Interaction with the pyridyl then led to a more stable
5-membered ring “A” as shown in 6. The Zn2+ cation in 6a could
further interact with the adjacent hydroxyl group to form the
fused 6-membered ring “B”, which requires twisting of the
“HBO” fragment from the Schiff base fragment (ring A). The
intermediate 7 could quickly lose a proton to give 4-Zn, which is
detected in the absorption spectrum. From the uorescence
titration data, the association constant was determined to be
images after addition of different metal ions to
EtOH : HEPES ¼ 1 : 1).
4 (10 mM in
which simultaneously induced both uorescence turn-on and a
large spectral shi.
1
In the H NMR of ligand 4 (Fig. 3), the signals at 10.56 and
10.02 ppm were attributed to the phenolic protons (ꢁOH0 and
–OH, respectively),18 while the signals at 11.09 ppm were
assigned to the amine.19 Upon addition of Zn2+ (one equiv.), the
–OH0 at 10.56 ppm was transformed to –OH00 (at ꢀ10 ppm), as a
consequence of forming 4-Zn. The unusually broad signals at
ꢀ10 ppm, when 0.2–0.5 equiv. of Zn2+ was added, were partially
attributed to the presence of both –OH (in 4) and –OH” (in
4-Zn). Two singlet ArꢁH signals (Hc or Hc' at 7.38, and Hd at 7.44
ppm) were gradually decreased with addition of Zn2+, in
agreement with the large electronic impact associated with the
formation of the metal complex. It should be noted that the
Ka ¼ 2.28 ꢂ 105 Mꢁ1
.
When Cd2+ was used, the interaction pattern appeared to be
1
quite different. The H NMR titration using Cd2+ revealed that
the pyridyl protons Ha and Hb were not affected aer addition of
one equiv. of Cd2+ cations (ESI Fig. S12†), indicating the lack of
tight and stable binding to the pyridyl ring. Therefore, the
equilibrium between 6b and 8 might occur without forming the
fused rings A–B in 4-Cd. This might be due to the larger size of
Fig. 3 1H NMR titration of 4 with Zn(OAc)2 in DMSO-d6; disappear-
ance of signal Hc at ꢀ7.38 ppm supports the 1 : 1 ligand-to-metal ratio. Fig. 4 Mass spectrum of 4-Zn (calcd for C20H14N4O3Zn: 422.0357).
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the Cd2+ cation (or longer Cd–N & Cd–O bonds), which desta-
bilizes the fused A–B rings in 4-Cd as observed from a molecular
modeling comparison (ESI Fig. S11†). The proposed fast equi-
librium between 6b and 8 accounts for the experimental nd-
ings: (a) addition of Cd2+ cations induced a smaller peak of
bathochromic shi in the absorption spectrum of 4 (Fig. 1),
indicating the weak binding of Cd2+ cations to phenol (without
removing phenolic protons) and (b) disappearance of phenolic
protons in 1H NMR (ESI Fig. S12†). Although the strength of
lewis acid (Zn2+ > Cd2+) could play a role in their differentiation,
Lewis acidity alone could not explain the lack of formation of
4-Cd under the experimental conditions (Scheme 3).
In the zinc-sensing process, an intriguing question is how
the zinc-binding in 4-Zn could affect the ESIPT event in HBO, in
comparison with the ligand 4. As shown in 1 / 2 (Scheme 1),
the ESIPT event in HBO is dependent on the electronic
connection between the phenol and benzoxazole fragments.
Computation with DFT at the B3LYP/6-31 G level revealed that
the LUMO of HBO in 4 had a large orbital interaction between
the phenol and benzoxazole fragments (Fig. 5), which could
facilitate the proton transfer in the excited state. The calculation
was consistent with the experimental nding that the ligand 4
gave only ESIPT emission, indicating the important role of the
electronic connection in the LUMO of HBO. The calculation
further revealed that the electronic connection between the
phenol and benzoxazole fragments became weaker in the
LUMO of 4-Zn, which might be responsible for partial conver-
sion of the excited 4-Zn (the enol tautomer) to its keto tautomer.
This led to emission from both enol and keto tautomers of 4-Zn.
In other words, the new zinc sensor exhibited several attractive
features: (1) using Schiff base binding to weaken ESIPT emis-
sion to reduce the background emission signal; Lack of enol
emission from 4 is also an improved feature over the previous
sensor 3;11 (2) introducing zinc binding to induce a large spec-
tral shi for emission in the NIR region; and (3) using zinc
binding to perturb the electronic linkage to attenuate the ESIPT,
thereby allowing dual emission.
Fig. 5 The HOMO and LUMO orbitals of 4-Zn and 4, calculated with
DFT at the B3LYP/6-31 G level using Gaussian 09. The double arrows
indicate the orbital lobes between the phenol and benzoxazole
fragments.
To illustrate the potential biological application, sensor 4
was applied to visualize intracellular Zn2+ in both human cancer
cells (HepG2) and human umbilical vein endothelial cells
(HUVECs) (see Fig. 6, and ESI Fig. S13† for more details). For
this purpose, cells were rst incubated with 30 mM of Zn2+ for
30 min and then further treated with 10 mM dye 4 for another
30 min before imaging. While very weak uorescence was
observed in cells treated with only dye 4 (Fig. 6b), strong
Fig. 6 Confocal fluorescence images of human mesenchymal stem
cells (hMSCs) excited with a 488 laser. The images were collected at
bright field (a and c) and green channel (535–565 nm; b and d) on an
Olympus FV1000-Filter confocal microscope. a / b: the cells were
incubated with dye 4 for 60 min at 37 ꢃC; c / d: the cells were first
treated with Zn(OAc)2 (30 mM) for 30 min and further exposed to dye 4
(10 mM) for another 60 min at 37 ꢃC.
Scheme 3 Possible formation of Zn2+ and Cd2+ complexes.
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uorescence was observed from both cell lines which are
treated with both Zn2+ and dye 4 (Fig. 6d and S13c†). These
results demonstrate that the probe is permeable to cells, binds
to intracellular Zn2+, and emits strong uorescent light upon
binding to the metal ion, and thus is highly suitable for deter-
mining intracellular Zn2+.
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In conclusion, we have demonstrated a cell permeable sensor
which is highly selective (almost specic) for zinc cations. The
sensor design successfully utilizes the Schiff base in the zinc-
binding event, which subsequently induces the ESIPT of HBO 4
to produce a large uorescence response. In contrast to the
existing sensors that typically give one signal, the new sensor
generates not only a large uorescence turn-on at ꢀ545 nm but
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Acknowledgements
This work was supported by National Institute of Health (Grant 18 The proton signal at 10.02 ppm is assigned to –OH near the
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ment from the University of Akron for partial support.
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2012 | J. Mater. Chem. B, 2014, 2, 2008–2012
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