Switchable and selective detection of Zn2+ or Cd2+ in living cells based on 3′-O-substituted arrangement of benzoxazole-derived fluorescent probes

Article abstract of DOI:10.1039/c4cc02335h

Two benzoxazole-derived ESIPT fluorescent sensors E1 and E2 show highly selective detection of Zn²⁺ and Cd²⁺, respectively, in aqueous solution and living cells. The selectivity switching from Zn ²⁺ to Cd²⁺ is a

Full text of DOI:10.1039/c4cc02335h

ChemComm
View Article Online
View Journal | View Issue
COMMUNICATION
Switchable and selective detection of Zn2+ or
Cd2+ in living cells based on 30-O-substituted
arrangement of benzoxazole-derived
fluorescent probes†
Cite this: Chem. Commun., 2014,
50, 7514
Received 29th March 2014,
Accepted 19th May 2014
Yongqian Xu,*a Liangliang Xiao,a Shiguo Sun,*a Zhichao Pei,a Yuxin Peia and
DOI: 10.1039/c4cc02335h
Yi Pangb
www.rsc.org/chemcomm
Two benzoxazole-derived ESIPT fluorescent sensors E1 and E2 identical spectral changes that make it difficult to discriminate
show highly selective detection of Zn2+ and Cd2+, respectively, in them. Thus it is a large challenge to design the sensors for highly
aqueous solution and living cells. The selectivity switching from selective detection of Zn2+ or Cd2+. Although many fluorescent
Zn2+ to Cd2+ is attributed to the different binding mode which is sensors with modified receptors can efficiently and highly detect
dependent on the 30-O-substituted arrangement.
Zn2+ or Cd2+, there is no reliable design guide for selective
recognition of Zn2+ or Cd2+. Recently, Xu et al. reported a
The zinc ion (Zn2+), as the second most abundant transition metal fluorescent sensor with a tautomerization-based transformable
ion in the human body, plays a very important role in biological receptor.10 Although the sensor shows distinct fluorescence
processes such as gene transcription, immune function, pathology, changes to discriminate between Zn2+ and Cd2+ because of
neural signal transmission, as well as catalysis of proteins.1 Zinc different binding modes (Zn2+ in an imidic acid form, Cd2+ in
imbalance is associated with a number of pathological disorders an amide tautomeric form), it can only recognize Zn2+ in the
including Alzheimer’s disease, epilepsy, Parkinson’s disease, presence of both Zn2+ and Cd2+ due to different binding affinity.
ischemic stroke and infantile diarrhea.2 On the other hand, the In order to study and understand the binding properties and
cadmium ion (Cd2+), being in the same group as the zinc ion in the diversity of Zn2+ and Cd2+, it is necessary to use a structure-tuned
periodic table, is a heavy metal and widely used in industry and receptor to ensure switchable selectivity for Zn2+ or Cd2+.
agriculture including the production of electroplating, metallurgy,
Recently, fluorescent sensors based on excited-state intramolecular
batteries, etc.3 Cd2+ is highly toxic and can be easily absorbed and proton transfer (ESIPT), as seen from 2-(20-hydroxyphenyl)benzoxazole
accumulated by plants and other organisms, leading to renal (HBO), have attracted more interest.7 ESIPT sensors exhibit dual
dysfunction, calcium metabolism disorders and cancers.3c Thereby emissions from both the excited enol and keto tautomers. Fluorescent
the measurement of Zn2+ or Cd2+ in physiological media has been sensing of metal ions could be realized by prohibiting ESIPT through
considered as an essential factor and the addressed target in these the coordination of metal ions with ESIPT centers, resulting in
relative diseases. Among the methods developed for Zn2+ or Cd2+ detectable spectral changes.7 Compared with the widely used photo-
sensing, fluorescent probes have attracted considerable attention induced electron transfer (PET) mechanism for Zn2+ or Cd2+, the
due to their simple operation, high sensitivity, noninvasiveness and fluorescent sensors based on ESIPT can afford many advantages
real time detection.4 Hitherto, significant effort has been devoted including dual fluorescence intensity changes and large Stokes shift.9
to the development of fluorescent sensors for Zn2+ or Cd2+ with They can detect metal ions with ratiometric fluorescent response and
successful applications to image in living cells.5 However, most of a near infrared (NIR) fluorescent signal (700–900 nm), providing the
them suffered from limited selectivity over biologically abundant self-calibration function and avoiding photodamage, scattering light
metal ions like Fe2+/Fe3+ and Cu2+ due to the moderate coordina- and strong interference derived from short wavelength emission in
tion nature of Zn2+ and the use of di-2-picolyamine (DPA) as a biological media.8
chelator.6 In addition, Zn2+ and Cd2+ have similar properties that
Herein, we present two ESIPT fluorescent sensors E1 and E2 for
lead to a similar binding mode to acceptors of sensors, providing Zn2+ and Cd2+ sensing, respectively (Scheme 1). Both of them show
high sensitivity and selectivity in aqueous solution. Compared with E1,
a College of Science, Northwest A&F University, Yangling, Shaanxi, P.R. China
E2 has no methyl group at the 30-hydroxyl position. With and without
the methyl group, the selectivity of the sensor switches from Zn2+ to
712100. E-mail: xuyq@nwsuaf.edu.cn, sunsg@nwsuaf.edu.cn
b Department of Chemistry & Maurice Morton Institute of Polymer Science,
Cd2+. As a proof-of-principle method, substituent arrangement-induced
The University of Akron, Akron, OH, 44325, USA
selectivity switching has been proved, which would be helpful to design
fluorescent sensors for efficient discrimination between Zn2+ and Cd2+.
† Electronic supplementary information (ESI) available: Details of the synthetic
procedure and additional spectra. See DOI: 10.1039/c4cc02335h
7514 | Chem. Commun., 2014, 50, 7514--7516
This journal is ©The Royal Society of Chemistry 2014

View Article Online
Communication
ChemComm
Scheme 1 Structure of E1 and E2.
The detailed synthetic procedure is described in the ESI.† Briefly, E1
was readily synthesized by condensation reaction between O-vanillin
and 2-aminophenol. Subsequently, E2 could be obtained from the
demethylation of E1 in the presence of BBr3. All these compounds were
fully characterized by 1H NMR, 13C NMR and HRMS.
Fig. 2 Emission intensity of E1 (10 mM) at 458 (a) and 880 nm (b) in
CH3CN/HEPES buffer (1 : 1, v/v, pH 7.4) in the presence of different metal
ions (200 mM) with the excitation at 360 nm (blank bar). Red bars represent
the intensity with subsequent addition of Zn2+ ions (200 mM).
In CH3CN/HEPES buffer (1 : 1, v/v, pH 7.4), E1 exhibited one
absorption peak at 325 nm, while upon addition of Zn2+, the
absorption at 325 nm gradually decreased, whereas a new absorp-
tion peak appeared at 370 nm with a well-defined isosbestic point at
340 nm (Fig. S1a, ESI†). Accordingly, upon excitation at 305 nm, the
maximum emission at 380 nm stabilized and a new emission peak
at 455 nm appeared, the intensity of which evidently increased upon
successive addition of Zn2+ (Fig. S2, ESI†). The two emission bands
at 380 and 455 nm can be attributed to the normal isomer (N*
emission) and tautomer (T* emission) of E1, respectively.7 The
observed fluorescence increase of the tautomer indicated that the
Zn2+ binding is beneficial for the stability of the tautomer. The new
emission peak can be used for the ratiometric fluorescent measure-
ment of Zn2+. Interestingly, upon excitation at 360 nm, except for an
emission band at 455 nm, a new NIR emission band at 880 nm
appeared (Fig. 1). Their fluorescence intensities gradually increased
linearly with addition of Zn2+ (Fig. S3, ESI†). After addition of
1 equivalent of Zn2+, the fluorescence quantum yield of E1 changes
from 0.0206 to 0.34. The Job’s plot revealed 1 : 1 stoichiometry for the
binding between E1 and Zn2+ (Fig. S4, ESI†). The binding constant
was calculated to be 6.51 ꢀ 104 Mꢁ1 with a detection limit of 1.63 ꢀ
10ꢁ8 M. The selectivity of E1 to various metal ions was further
examined. As shown in Fig. S6 (ESI†), only Zn2+ promotes significant
fluorescence intensity enhancement at 458 and 880 nm, whereas
other metal ions cause no detectable spectral change except that
Cu2+ induces somewhat fluorescence quenching. To explore the
possible utility of E1 as a fluorescent sensor for Zn2+, competitive
experiments were carried out in the presence of 20 equivalents of
Zn2+ and 20 equivalents of various other cations (Fig. 2). Although
Cd2+ exerts a weak increasing effect on the probe, the little inter-
ference can be eliminated by cysteine (Cys) that is abundant
in vivo.10 The E1 + Zn2+ complex is stable in the presence of Cys
while the E1 + Cd2+ complex is dissociated owing to the competition
of Cys (Fig. S7, ESI†). These results suggested that E1 shows excellent
binding selectivity for Zn2+ and can detect Zn2+ with NIR emission.
Compared with E1, E2 only lost the methyl group at the
30-hydroxyphenyl position. Under identical conditions, E2 shows
similar UV-Vis spectral change upon addition of Cd2+ with a well-
defined isosbestic point at 350 nm (Fig. S1b, ESI†). Upon excitation
at 305 nm, E2 has two maximum emission bands at 360 and 468 nm
(Fig. S8, ESI†), assigned to the normal isomer (N* emission) and
tautomer (T* emission), respectively. When excited at 360 nm, the
fluorescence intensity at 468 nm evidently increased with successive
addition of Cd2+. The fluorescence quantum yield (F) increased
from 0.0012 to 0.032 in the presence of 1 equivalent of Cd2+. Its
fluorescence intensity gradually increased linearly with addition of
Cd2+ (Fig. 3a and Fig. S9, ESI†). The Job’s plot revealed 1 : 1
stoichiometry for the binding between E2 and Cd2+ (Fig. S10, ESI†).
The binding constant was calculated to be 1.99 ꢀ 104 Mꢁ1 and the
detection limit was 1.33 ꢀ 10ꢁ7 M. In sharp contrast to E1,
E2 showed excellent selectivity to Cd2+. Competitive experiments
were carried out in the presence of 20 equivalents of Cd2+ and
20 equivalents of various other cations (Fig. 3b). Except that
Mg2+ induced a little fluorescence increase, other various metal ions
including Zn2+ ions caused no detectable spectral change at 469 nm.
The result suggested that E2 can highly detect Cd2+ without inter-
ference by Zn2+. Moreover, competitive experiments were carried out
in the presence of Cys, showing that there is no interference from
Cys for probe E2 to detect Cd2+ ions possibly due to the stronger
binding capability between E2 and Cd2+ (Fig. S21 and S22, ESI†).
Both of them are pH independent in the range of 7–8, demon-
strating that they can detect metal ions in biological environ-
ments (Fig. S11 and S12, ESI†).
Fig. 3 (a) Fluorescence emission spectra of E2 (10 mM) upon addition of
Cd2+ in CH3CN/HEPES buffer (1 : 1, v/v, pH 7.4), lex = 360 nm; (b) emission
intensity of E2 (10 mM) at 469 nm in CH3CN/HEPES buffer (1 : 1, v/v, pH 7.4)
in the presence of different metal ions (200 mM) with the excitation at
360 nm (blank bar). Red bars represent the intensity with subsequent
addition of Cd2+ ions (200 mM).
Fig. 1 The fluorescent spectral change of E1 (10 mM) in short and
long wavelength regions upon addition of Zn2+ in CH3CN/HEPES buffer
(1 : 1, v/v, pH 7.4), lex = 360 nm.
This journal is ©The Royal Society of Chemistry 2014
Chem. Commun., 2014, 50, 7514--7516 | 7515

View Article Online
ChemComm
Communication
Interestingly, the selectivity can be switched from Zn2+ to Cd2+ efficient fluorescent detection in living cells (Fig. 4 and Fig. S17,
through a facial substituent effect of benzoxazole derivatives. To ESI†). Moreover, NIR red emission can be detected in the case of
further evaluate the response nature and gain insight into the E1 treated with Zn2+.
1
recognition mechanism for Zn2+ and Cd2+, the H NMR titration
In summary, two kinds of benzoxazole-derived ligands E1 and
spectra of E1 with Zn2+ and E2 with Cd2+ were investigated. The E2, being different at a methyl substituent, have been presented.
chemical shift of the hydroxyl –OH can be used to value whether the For E1, it can selectively detect Zn2+ in buffer solution and living
metal ion is bound to the hydroxyl oxygen. For E1, the Zn–O bond cells with fluorescence intensity increasing at 455 and 880 nm. The
results in the disappearance of the –OH resonance peak at 11.20 selectivity can be further improved without interference from Cd2+
with addition of 2 equivalents of Zn2+ in DMSO-d6 (Fig. S13, ESI†). in the presence of biological Cys. For E2, it shows excellent
This result indicates that the –OH group was involved in the binding selectivity toward Cd2+ and can be applied for living cell imaging.
with Zn2+. In the case of E2, the Cd2+ binding results in the upfield The possible binding modes between them were investigated by
shift of the –OH proton at the 20 position from 11.10 to 11.07 and 1H NMR titration spectra, from which the reasons and recognition
the downfield shift of the –OH proton at the 30 position from 9.56 to mechanisms were interpreted. As a proof-of-principle method,
9.60 (Fig. S14, ESI†). The two different effects on the –OH proton substituent arrangement-induced selectivity switching would be
could be considered as a result of the Cd2+ binding. Through-bond helpful in the design of fluorescent sensors for other metal ions.
propagation increases the electron density on the hydroxyl group at
This work was supported by the National Natural Science
the 20 position and produces a shielding effect. While a through- Foundation of China (Grant No. 21206137), the Scientific
space effect increases the polarization of the hydroxyl group at the 30 Research Foundation of Northwest A&F University (Z111021103
position, the partial positive charge causes a deshielding effect and and Z111021107) and the Fundamental Research Funds for the
downfield shift of its proton.11 The non-vanishing of –OH protons at Central Universities (2452013py014).
20 and 30 positions after addition of Cd2+ suggested that the two
hydroxyl groups do not participate in the binding with Cd2+. The
strong intramolecular hydrogen bonding possibly prevents Cd2+ ions
from binding with hydroxyl groups. The proposed binding modes of
E1 with Zn2+ and E2 with Cd2+ were observed, from which different
ion-induced binding profiles are attributed to different selectivity
(Scheme S1, ESI†). More direct evidence was obtained from the ESI
mass spectra, where the ion peak at m/z 510.31 (Fig. S15, ESI†)
corresponded to the molecular ion peak of [E1–H + Zn2+ + 2CH3OH +
H2O + ClO4ꢁ + Na+] (calcd = 510.15). For E2, the peak at m/z 451.74
(Fig. S16, ESI†) corresponded to the molecular ion peak of [E2 +
Cd2+ + CH3OH + H2O + NO3ꢁ] (calcd = 451.69).
To further investigate the biological application of E1 and E2,
a fluorescence microscopy experiment in living cells was carried
out. When ovarian cancer cells (SKOV-3) were incubated with
10 mM E1 and E2 in culture medium at 37 1C for 1 h, relatively,
no detectable emission was observed. After incubation with Zn2+
and Cd2+ for E1 and E2, respectively, strong green emission can
been clearly seen, indicating a very good cellular uptake and
Notes and references
1 (a) C. J. Frederickson, J. Y. Koh and A. I. Bush, Nat. Rev. Neurosci.,
2005, 6, 449; (b) J. M. Berg and Y. Shi, Science, 1996, 271, 1081;
(c) M. Lu and D. Fu, Science, 2007, 317, 1746.
2 (a) A. I. Bush, Curr. Opin. Chem. Biol., 2000, 4, 184; (b) C. F. Walk and
R. E. Black, Annu. Rev. Nutr., 2004, 24, 255.
3 (a) S. Satarug and M. R. Moore, Environ. Health Perspect., 2004,
112, 1099; (b) M. Waisberg, P. Joseph and B. Hale, Toxicology, 2003,
192, 95; (c) R. A. Goyer, J. Liu and M. P. Waalkes, Biometals, 2004, 17, 555.
4 (a) Z. Xu, K.-H. Baek, H. N. Kim, J. Cui, X. Qian, D. R. Spring, I. Shin
and J. Yoon, J. Am. Chem. Soc., 2010, 132, 601; (b) A. P. de Silva,
H. Q. N. Gunaratne, T. Gunnlaugsson, A. J. M. Huxley, C. P. McCoy,
J. T. Rademacher and T. E. Rice, Chem. Rev., 1997, 97, 1515;
(c) W. Xuan, C. Chen, Y. Cao, W. He, W. Jiang, K. Liu and W. Wang,
Chem. Commun., 2012, 48, 7292–7294; (d) M. Collot, C. Loukou,
A. V. Yakovlev, C. D. Wilms, D. Li, A. Evrard, A. Zamaleeva,
L. Bourdieu, J. F. Leger, N. Ropert, J. Eilers, M. Oheim, A. Feltz and
J. M. Mallet, J. Am. Chem. Soc., 2012, 134, 14923–14931; (e) Z. Xu, X. Liu,
J. Pan and D. R. Spring, Chem. Commun., 2012, 48, 4764.
5 (a) C. Lu, Z. Xu, J. Cui, R. Zhang and X. Qian, J. Org. Chem., 2007,
72, 3554; (b) L. Xue, C. Liu and H. Jiang, Org. Lett., 2009, 11, 1655–1658.
6 (a) X. Zhang, K. S. Lovejoy, A. Jasanoff and S. J. Lippard, Proc. Natl.
Acad. Sci. U. S. A., 2007, 104, 10780; (b) B. A. Wong, S. Friedle and
S. J. Lippard, J. Am. Chem. Soc., 2009, 131, 7142; (c) X. Zhang,
D. Hayes, S. J. Smith, S. Friedle and S. J. Lippard, J. Am. Chem.
Soc., 2008, 130, 15788; (d) E. Tomat, E. M. Nolan, J. Jaworski and
S. J. Lippard, J. Am. Chem. Soc., 2008, 130, 15776; (e) J. E. Kwon,
S. Lee, Y. You, K.-H. Baek, K. Ohkubo, J. Cho, S. Fukuzumi, I. Shin,
S. Y. Park and W. Nam, Inorg. Chem., 2012, 51, 8760; ( f ) Y. You,
S. Lee, T. Kim, K. Ohkubo, W.-S. Chae, S. Fukuzumi, G.-J. Jhon,
W. Nam and S. J. Lippard, J. Am. Chem. Soc., 2011, 133, 18328;
(g) Y. Wu, X. Peng, B. Guo, J. Fan, Z. Zhang, J. Wang, A. Cui and
Y. Gao, Org. Biomol. Chem., 2005, 3, 1387.
7 (a) J. Wu, W. Liu, J. Ge, H. Zhang and P. Wang, Chem. Soc. Rev., 2011,
40, 3483; (b) T. Mutai, H. Sawatani, T. Shida, H. Shono and K. Araki,
J. Org. Chem., 2013, 78, 2482.
8 W. Chen, B. D. Wright and Y. Pang, Chem. Commun., 2012, 48, 3824.
9 R. Y. Tsien, in In Fluorescent and Photochemical Probes of Dynamic
Biochemical Signals inside Living Cells, ed. A. W. Czarnik, American
Chemical Society, Washington, DC, 1993, pp. 130–146.
10 (a) M. E. McMenamin, J. Himmelfarb and T. D. Nolin, J. Chromatogr.
B: Anal. Technol. Biomed. Life Sci., 2009, 877, 3274–3281; (b) C. Hwang,
A. J. Sinskey and H. F. Lodish, Science, 1992, 257, 1496–1502.
11 D. E. Gomez, L. Fabbrizzi, M. Licchelli and E. Monzani, Org. Biomol.
Chem., 2005, 3, 1495–1500.
Fig. 4 Fluorescence images of SKOV-3 cells. (a–d) SKOV-3 cells incubated
with probe E1 (10 mM) for 30 min; (e–h) images of cells after treatment with
probe E1 (10 mM) for 30 min and subsequent treatment of the cells with 50 mM
Zn2+ for 20 min. (a and e) Bright-field images of the SKOV-3 cells in samples;
(b and f) images taken in green field; (c and g) images taken in red field; and
(d and h) the overlap of brightfield and fluorescence. Scale bar: 20 mm.
7516 | Chem. Commun., 2014, 50, 7514--7516
This journal is ©The Royal Society of Chemistry 2014