A water-soluble, small molecular fluorescence probe based on 2-(2′-hydroxyphenyl) benzoxazole for Zn2+ in plants

Article abstract of DOI:10.1016/j.tetlet.2018.09.057

A water-soluble, small molecular zinc fluorescence probe (ZFP) based on 2-(2′-hydroxyphenyl) benzoxazole was prepared. It exhibited high selectivity and sensitivity to Zn²⁺ than the other metal ions. The highest fluorescence enhancement was obs

Full text of DOI:10.1016/j.tetlet.2018.09.057

Accepted Manuscript
A water-soluble, small molecular fluorescence probe based on 2-(2’-hydroxy-
2
+
phenyl) benzoxazole for Zn in plants
Yuting Shang, Shuwen Zheng, Madalitso Tsakama, Miao Wang, Weihua Chen
PII:
S0040-4039(18)31159-6
https://doi.org/10.1016/j.tetlet.2018.09.057
TETL 50296
DOI:
Reference:
To appear in:
Tetrahedron Letters
Received Date:
Revised Date:
Accepted Date:
19 July 2018
13 September 2018
22 September 2018
Please cite this article as: Shang, Y., Zheng, S., Tsakama, M., Wang, M., Chen, W., A water-soluble, small molecular
2
+
fluorescence probe based on 2-(2’-hydroxyphenyl) benzoxazole for Zn in plants, Tetrahedron Letters (2018), doi:
https://doi.org/10.1016/j.tetlet.2018.09.057
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Graphical Abstract
.
A water-soluble, small molecular
Leave this area blank for abstract info.
fluorescence probe based on 2-(2’-
hydroxyphenyl) benzoxazole for Zn in
plants
2
+
1
1
Yuting Shang† , Shuwen Zheng† ,
1
1
*1,2
Madalitso Tsakama , Miao Wang and Weihua Chen
O
CH2OH
O
N
HMTA
O
N
NaBH4
2
O
N
CF3COOH, reflux
CH Cl2
CH OH
OH
OH
O
OH
2
1
2
ZFP
4
51nm
Blank
1 eq Zn
2
+
800
600
400
200
0
2
3
4
5
6
7
8
9
2+
ZFP
ZFP+Zn
1
0
471nm
350
400
450
500
550
600
Wavelength (nm)

1
Tetrahedron Letters
A water-soluble, small molecular fluorescence probe based on 2-(2’-hydroxyphenyl)
2+
benzoxazole for Zn in plants
1
1
1
1
1,2
Yuting Shang† , Shuwen Zheng† , Madalitso Tsakama , Miao Wang and Weihua Chen
1
Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences, Beijing 100193
Key Laboratory of Agro-products Quality and Safety Control in Storage and Transport Process, Ministry of Agriculture, P.R. China
2
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A water-soluble, small molecular zinc fluorescence probe (ZFP) based on 2-(2’-hydroxyphenyl)
2+
benzoxazole was prepared. It exhibited high selectivity and sensitivity to Zn than the other
2+
metal ions. The highest fluorescence enhancement was observed in the presence of Zn owing
to the inhibition of excited-state intramolecular proton transfer (ESIPT). Furthermore,
2+
Available online
fluorescence imaging experiments confirmed that ZFP can be used to monitor Zn in
biological systems.
Keywords:
Fluorescence
2
009 Elsevier Ltd. All rights reserved.
2
-(2’-hydroxyphenyl) benzoxazole
2
+
Zn
ESIPT
Plants
Metal-induced fluorescence enhancement probe is
a
resonance energy transfer (FRET) [16], twisted intramolecular
charge transfer (TICT) [17], excimer formation [18] and imine
isomerization [19]. However, these mechanisms have some
limitations. For example, the emission signal from ICT is often
pH-dependent, while PET-based probe requires a relatively larger
structure as an electron-donor group is attached to the
potential tool that can be used in fluorescence microscopy to
explore biological events which include: location, concentration
of metals and change in their population, in responding to
cellular events [1]. The high cell permeability, low cytotoxicity
and high sensitivity and selectivity for a particular metal ion are
prerequisites of a probe can be successfully used in biological
systems [2].
Zinc is an indispensable trace element which plays an
important role in biological systems [3]. It is involved in many
processes such as neurotransmission [4]. enzyme regulation [5],
gene expression [6] and cell apoptosis [7]. Plants also require the
proper balance of zinc for normal growth and optimum yield.
Zinc is usually transported from the root to xylem through
epidermis, cortex and endodermis. In the xylem, it moves as a
complexed form, which makes it more easily to influx into the
phloem and embryoid. Evaluating the severity of zinc deficiency
symptoms on leaves as well as monitoring shoot and seed zinc
concentration and content appear to be useful approaches for
screening large populations for zinc efficiency [8]. Detection of
zinc is, therefore, of fundamental importance in understanding
the impact of zinc in the related biological systems [9].
fluorophore with
a
proper distance [20]. Excited-state
intramolecular proton transfer (ESIPT) has emerged to be an
attractive mechanism for probe design, due to its capability to
generate emission with a large Stokes shift [21]. Furthermore, it
also has been well established as a design strategy for ratiometric
[22] or two-photon fluorescent probes [23].
2-(2’-hydroxyphenyl)benzoxazole (HBO) is a common
chromophore that exhibits the characteristic ESIPT emission.
However, HBO tends to form aggregate in polar solvents such as
in H O and MeOH [24]. which hampers its application. Herein,
2
we reported a novel HBO-based zinc fluorescence probe (ZFP)
which has two hydoxymethyl groups on the phenol ring to
improve the water solubility (Fig.1). The added –CH
2
OH group
could also be a reactive site to perturb the metal ion binding. The
2+
results show that ZFP exhibited a high selectivity in binding Zn ,
which was accompanied with a large chelation-enhanced
fluorescence.
Over the past decade, several fluorescent probes for zinc
sensing have been developed by using different fluorophores,
such as benzoresorufin [10], Rhodol [11] and BODIPY dyes [12].
They are generally based on the following sensing mechanisms
The desired ZFP probe was synthesized conveniently by
reduction of 2, which was readily available from HBO 1 by using
the previously reported procedure [25]. Crystal structure (Fig.2)
revealed that ZFP could exist in two possible rotamers, which has
been observed in HBO [26] but not in HBO derivatives.
[
13] that include intramolecular charge transfer (ICT) [14],
photoinduced electron transfer (PET) [15], fluorescence
—
——

Correspond author: Prof. Chen, chenweihua@cass.cn
These authors contributed equally to this work
†

2
The probe exhibited two absorption peaks, with a minor
peak at 295 nm and a major peak at 322 nm, corresponding to the
π-π* transitions respectively. When excited at 320 nm, ZFP
displayed a maximum emission peak at 471 nm, which can be
attributed to the emission from the keto tautomer that was
generated from the intramolecular hydrogen-bonded enol via the
fast ESIPT process.
4
49 nm
Blank
8
00
+
K
+
Na
2
+
+
Ca
Ba
Mg
2
600
400
2
+
3+
Al
Fe
Cu
Cd
High selectivity is extremely important for a probe. To
3
+
2+
evaluate the selectivity of ZFP towards Zn , the fluorescence
2+
2+
+
+
response behaviors of ZFP towards various metal ions (K , Na ,
2+
2+
2+
3+
3+
2+
2+
471 nm
57 nm
2+
Ca , Ba , Mg , Al , Fe , Cu and Cd ) were investigated in
Zn
4
200
HEPES buffer solution. As shown in Fig.3a, only the addition of
2+
Zn caused a prominent emission enhancement, whereas the
other common metal ions did not cause any obvious changes.
0
2+
Interestingly, the Cd binding only caused a slight spectral blue
shift to ~457 nm without inducing the fluorescence enhancement.
Based on the use of a UV lamp, the fluorescence color changed
3
50
400
450
500
550
600
Wavelength (nm)
2+
from light blue to brilliant blue only in the presence of Zn
Fig.3a inset and Fig.S1), which could easily be detected by the
naked-eye. To determine the possible interference of the other
(a)
(
2+
metal ions on probe ZFP for Zn , competitive binding
ZFP+metal
ZFP+metal+Zn
8
7
6
5
4
3
2
1
0
2+
experiments were performed. As shown in Fig.3b, other metal
2+
ions (except Cu ) had small or no obvious interference with the
2+
2+
detection of Zn . ZFP with Cu shown fluorescence quenching,
it may be due to paramagnetic fluorescence quenching. In ZFP-
2+
Cu complex, the formally forbidden intersystem crossing (ISC)
7
became faster when the presence of a d Cu (II) ion [27].
2+
2+
Furthermore, Cd is known as a common competitor for Zn ,
but it did not cause a notable change in fluorescence intensity.
2+
2+
Subsequent addition of Zn to ZFP-Cd , however, generated a
sizable fluorescence enhancement. The results suggested that
2+
2+
ZFP could be a good probe for Zn . Low interference from Cd
binding could be due to the presence of two adjacent hydroxyl
2+
+
+
2+
2+
2+
3+
3+
2+
2+
groups, i.e. 2’-hydroxy and 3’-hydroxymethyl groups, which
Zn
K
Na Ca Ba Mg Al Fe Cd Cu
2+
could have strong binding to Cd as observed in a similar system
28].
[
2+
(b)
The fluorescence titration of ZFP with Zn was performed
to further investigate the chemosensing properties of ZFP. As
shown in Fig.4a, the weak emission at 471 nm had a
hypochromatic shift to 451 nm and the intensity was greatly
Fig.3 (a) Fluorescence emission spectra of ZFP (10 µM) in the
presence of 10 equiv of various metal ions; Inset: the color
change of ZFP in the presence of Zn ; (b) Metal ions selectivity
2+
2+
enhanced upon addition of Zn . The fluorescence quantum yield
of ZFP. Bars represented the fluorescence ratio F451/F . Black
0
2
+
(
Φ) increased from 0.012 to 0.131 in the presence of Zn . The
bars: ZFP (10 µM) with various metal ions (100 µM). Red bars:
3
-1
association constant K
d
was calculated to be 1.19×10 M for
2+
ZFP (10 µM) with various metal ions (100 µM) and Zn (100
2+
2+
ZFP-Zn which indicated the strong affinity of ZFP to Zn , with
µM). All data were obtained in HEPES buffer (20 mM, pH 7.0),
a detection limit of 78 nM. In addition, fluorescence intensity
λex=320 nm.
2+
increased linearly with the Zn concentration in the range 0–10
µM (Fig.4b), which means that ZFP is suitable for the
quantitative detection of Zn .
2+
451nm
Blank
2+
800
1 eq Zn
2
3
4
5
O
O
CH2OH
CH2OH
O
N
HMTA
3
O
N
NaBH4
CH Cl
O
N
600
CF COOH, reflux
2
2
OH
OH
OH
6
7
8
9
10
1
2
ZFP
4
00
00
0
Fig.1 Synthesis scheme of ZFP
2
471nm
350
400
450
500
550
600
Wavelength (nm)
(a)
Fig.2 Crystal structure of ZFP

3
2
+
The potential application of ZFP for Zn in biological
samples was examined by using fluorescence imaging
Equation
Weight
y = a + b*x
No Weighting
232.54696
2+
experiments. HepG2 cells were first incubated with Zn
200
180
160
140
120
100
Residual Sum of
Squares
followed by ZFP. It was found that zinc-treated cells exhibited
strong blue fluorescence, while cells incubated with ZFP alone
displayed a very weak intracellular fluorescence. Incubation with
EDTA reduced the fluorescence intensity, indicating that the
fluorescence signals were a consequence of the response to
Pearson's r
0.9848
Adj. R-Square
0.96607
Value
83.48985
9.51993
Standard Error
3.6831
0.59359
B
B
Intercept
Slope
2+
intracellular Zn (Fig.6).
With the success in vitro imaging, we became interested in
knowing the in vivo imaging potential of ZFP. White Dianthus
flowers were used for studying this ability. With time, flower
filaments showed fluorescence which was easily observed and
zinc-treated flowers exhibited stronger fluorescence than flowers
treated with the probe alone (Fig.7). The movement of ZFP and
0
2
4
6
8
10
2+
the translocation of Zn were studied by the petal of White
Concentration ()
Dianthus flowers (Fig.8). These imaging experiments supported
2+
the possibility of using ZFP in fluorescence imaging of Zn in
biological systems in real time. Animal models have always been
used in vivo imaging experiments, but they are expensive and
associated with lengthy processes. So, this approach may be a
guidance in screening in vivo imaging potential of a probe before
being tested on animals. Furthermore, a cytotoxicity test was
performed to evaluate toxic effects of our probe. This
experimental result demonstrated that ZFP had low toxicity to the
cells (Fig.S10). All these results had shown that ZFP was cell-
(
b)
Fig.4 (a) Fluorescence emission spectra changes of ZFP (10 µM)
in the presence of different equiv of Zn . All data were obtained
in HEPES buffer (20 mM, pH 7.0), λex=320 nm; (b) The plot of
fluorescence intensity at 451 nm versus the concentration of Zn
2+
2+
added.
UV-vis absorption of ZFP showed a major peak at 322 nm
Fig.5). Upon addition of Zn , the absorption band at 322 nm
2+
permeable and capable of imaging of Zn in biological systems.
2
+
(
decreased, which was accompanied with the formation of a new
band at 362 nm. An isosbestic point was observed at 342 nm,
indicating that the formation of a new species as free ZFP ligand
(a)
(b)
(c)
2+
was converted to its Zn complex. A Job’s plot indicated a 1:1
2+
complexation stoichiometry between ZFP and Zn (Fig.S3), and
this was further confirmed by the appearance of a peak at
2+
+
m/z=335.0, which was assigned to [ZFP+Zn -H] in mass
spectrum (Fig.S17). The results verified that the experimental
isotope pattern matches the theoretical isotope pattern .
Fig.6 Fluorescence images of HepG2 cells (λex=405 nm). (a)
Blank
Cells were incubated with 10 µM ZFP for 1 h; (b) Cells were
2+
1
2
3
4
5
6
7
8
9
1
1
2
eq Zn
0.10
0.08
0.06
0.04
0.02
0.00
treated with 20 µM ZnCl
incubated with 10 µM ZFP for 1 h; (c) Cells were pretreated with
20 µM ZnCl /sodium pyrithione and 10 µM ZFP for 1 h, then
2
/sodium pyrithione for 1 h and then
2
3
22 nm
incubated with 20 µM EDTA for 1 h.
0
5
0
3
62 nm
(a)
25
30
35
40
45
2
95 nm
50
(b)
(c)
300
350
400
Wavelength (nm)
Fig.5 UV-vis absorption spectra changes of ZFP (10 µM) in the
2+
presence of different equiv of Zn in HEPES buffer (20 mM, pH
7.0), λex=320 nm.
Fig.7 Fluorescence images of White Dianthus flowers. (a)
Flowers were observed in the light before treated; (b) Flowers
were observed under the UV lamp before treated; (c) Flowers
were observed under the UV lamp after treated 1h. Flowers from
To further investigate the stability of ZFP, the effect of pH
on ZFP was examined. ZFP was stable within the pH=1.05 to
2+
8
.00, and the fluorescence intensity of ZFP-Zn showed a
significant response at pH=7.5, meaning the ESIPT turn-off
2+
2+
left to right: one was only incubated with 1 mM Zn for 1 h, one
could be accomplished by Zn (Fig.S4). In addition, EDTA
ethylene diamine tetraacetic acid, a zinc ion chelator) test
2+
was treated with 1 mM Zn for 1 h and then incubated with 500
µM ZFP for 1 h and the other one was only incubated with 500
µM ZFP for 1 h.
(
indicated that ZFP could be used as a reversible fluorescence
probe in buffer solution (Fig.S5). These results suggested that
ZFP had the potential to be applied in the biological field.

4
(a)
(b)
(c)
(d)
References and notes
[
(
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1
[
(g)
(h)
[
4
[
(
(2018) 1133-1136.
[15] (a) Chantalakana, K.; Choengchan, N.; Yingyuad, P.;
Fig.8 Fluorescence images of a piece of petals (λex=405 nm). (a)
Petal was only incubated with 1 mM Zn for 1 h; (b) Petal was
Thongyoo, P., Tetrahedron Lett 57 (2016) 1146-1149;
(b) Tateno, K.; Ogawa, R.; Sakamoto, R.; Tsuchiya, M.; Otani, T.;
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(c) Outlaw, V. K.; Zhou, J.; Bragg, A. E.; Townsend, C. A., Rsc
Adv 6 (2016) 61249-61253;
2+
only incubated with 500 µM ZFP for 1 h; (c) Petal was treated
2+
with 1 mM Zn for 1 h and then incubated with 500 µM ZFP for
2+
1
h; (d) Petal was treated with 1 mM Zn for 1 h and then
incubated with 500 µM ZFP for 2 h; (e) and (f) were the upper
(d) Zhang, W.; Tang, B.; Liu, X.; Liu, Y.; Xu, K.; Ma, J.; Tong, L.;
Yang, G., Analyst 134 (2009) 367-371.
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Chem 82 (2010) 3108-3113.
and latter half of (b) only ZFP treated, (g) and (h) were the upper
2+
and latter half of (d) Zn and ZFP treated (observed under
fluorescence microscope).
In summary, we designed
benzoxazole based zinc fluorescence probe ZFP that had simple
a
2-(2’-hydroxyphenyl)
[17] Shi, B.; Zhang, Y.; Wei, T.; Lin, Q.; Yao, H.; Zhang, P.; You,
X., Sensor Actuat B-Chem 190 (2014) 555-561.
construction and good water solubility. Notably, ZFP showed
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K.; Townsend, C. A.; Bragg, A. E., Chemistry: A European
Journal 22 (2016) 15212-15215.
2+
high selectivity and sensitivity for Zn over the other metals in
2+
aqueous solution. Zn induced a dramatic enhancement in
fluorescence intensity and blue-shifted in wavelength of ZFP
through the inhibition of ESIPT. A linear fluorescence intensity
2+
response to the concentration of Zn suggested that ZFP could
2+
qualitatively and quantitatively monitor Zn concentration.
2+
Furthermore, ZFP could successfully be used to detect Zn in
biological systems. Therefore this zinc fluorescent probe has
great potential to be applied in chemical and biological fields.
Acknowledgments
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We are grateful for the financial support from the project of
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“
Source Identification and Contamination Characteristics of
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(
Development Program of China. The authors also wish to thank
the Collaborative Innovation Task of CAAS (CAAS-
XTCX2016005) for financial support.
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in the online version, at http://
[

5
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6




Reliable synthesis of the probe ZFP whose structure
was confirmed by X-ray analysis
The probe shows high sensitivity and selectivity
toward zinc ions against other metal ions
Unique sensing mechanism with perturbation of of
enol/keto tautomer emission
Excellent biocompatibility to ensure the probe to
easily transport in both xylem and phloem of plants