Six-membered spirocycle triggered probe for visualizing Hg2+ in living cells and bacteria-EPS-mineral aggregates

Article abstract of DOI:10.1021/ol401795m

A novel rhodamine based probe with a unique six-membered spirocycle was rationally designed for detection of Hg²⁺ with greatly improved selectivity, sensitivity, and photostability. The probe has been shown to be suitable for Hg²⁺ imaging in living cells and mapping Hg²⁺ distribution in living cell-EPS-mineral aggregates under anoxic conditions.

Full text of DOI:10.1021/ol401795m

ORGANIC
LETTERS
2013
Vol. 15, No. 17
4334–4337
Six-Membered Spirocycle Triggered
Probe for Visualizing Hg2þ in Living Cells
and BacteriaꢀEPSꢀMineral Aggregates
Zheng Yang,†, Likai Hao,‡, Bing Yin,†,§ Mengyao She,† Martin Obst,‡
Andreas Kappler,‡ and Jianli Li*,†
Ministry of Education Key Laboratory of Synthetic and Natural Functional Molecule
Chemistry, College of Chemistry & Materials Science, Northwest University, Xi’an,
Shaanxi 710069, P. R. China, Center for Applied Geoscience, Institute for Geoscience,
Eberhard Karls University Tuebingen, Hoelderlinstr. 12, Tuebingen 72074, Germany,
and College of Chemistry, Beijing Normal University, No. 19, XinJieKouWai Street,
HaiDian District, Beijing 100875, P. R. China
lijianli@nwu.edu.cn
Received June 25, 2013
ABSTRACT
A novel rhodamine based probe with a unique six-membered spirocycle was rationally designed for detection of Hg2þ with greatly improved
selectivity, sensitivity, and photostability. The probe has been shown to be suitable for Hg2þ imaging in living cells and mapping Hg2þ distribution
in living cellꢀEPSꢀmineral aggregates under anoxic conditions.
Mercury has been known as one of the most ubiquitous
and hazardous pollutants in nature.1 Methods are required
for mapping and quantifying Hg2þ in biological samples.
Fluorescence staining has advantages of high sensitiv-
ity and spatial resolution in combination with, in micro-
scopy, being nondestructive to the samples and less cell-
damaging.2 By virtue of the desirable properties,3 excellent
examples of rhodamine based fluorescent probes have
been developed for biologically relevant metal species on
the basis of an ion-induced ring-opening process.4
Reported Hg2þ probes usually contain a five-membered
spirolactam moiety.5 In general, an appropriate ligand at
the spirolactam ring can induce color and fluorescence
changes by activating the carbonyl group upon binding of
metal ions. However, the application of these probes is
limited by weak fluorescence intensity, short fluorescence
stability, and even fluorescence quenching.6 These draw-
backs can potentially be addressed by expanding beyond a
† Northwest University.
‡ Eberhard Karls University Tuebingen.
§ Beijing Normal University.
Z.Y. and L.K.H. contributed equally to this work.
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r
10.1021/ol401795m
Published on Web 08/12/2013
2013 American Chemical Society

five-membered spirocycle. At present, the development of
highly sensitive and clinically applicable diagnostic meth-
ods for Hg2þ has attracted great attention.7
Among the most attractive methodological innovations,
expansion of the spirocycle has the potential to improve
the stability of the probe while providing more binding
positions, thus potentially improving the selectivity and
fluorescent properties.8 Although our group and others
have devoted much effort toward enlarging the spirocycle
to a six-member moiety, few satisfying products have been
demonstrated to date.
Figure 2. Fluorescence intensity (a) and absorption (b) changes
of 1 (20 μM) upon addition of Hg2þ (0ꢀ1.5 equiv) in ethanolꢀ
PBS (4/6, v/v, pH 7.4) solution (excitation at 550 nm).
30 nM. About 1.0 equiv of Hg2þ was required until a
plateau was reached with the fluorescence intensity in-
creased for more than 1000-fold. Under these conditions, a
linear correlation between 1 and the concentration of Hg2þ
ranged from 30 nM to 20 μM, indicating the suitability for
quantitative determination of Hg2þ (Figure S3).
The fluorescence intensity in the presence of Hg2þ was
compared to other metals to study the selectivity of 1 and
the competition caused by other ions (Figure 3). The probe
showed excellent selectivity to Hg2þ with dramatic absor-
bance and fluorescence changes, whereas other metal ions
showed insignificant responses. Only Agþ had a slight ef-
fect which is significantly inferior and easily quenched. Var-
iation of the Hg2þ counterion had negligible effects on the
fluorescence intensity. The competition experiments which
were carried out by adding Hg2þ to the 1 solution in the
presence of other metalions revealedthat the selectivity of1
to Hg2þ did not significantly experience interference from
the commonly coexistent ions, although Agþ, Cu2þ, and
Fe3þ induced slight fluorescent quenching or enhancement.
Figure 1. Synthesis of rhodamine based probe 1.
Herein we describe the design and application of a
hydrazinobenzothiazole based six-membered probe 1
(Figure 1). The nonplanar six-membered spirocycle in 1
has been proven to be well suited for the recognition of
Hg2þ with improved optical properties relative to reported
probes.6,9 Furthermore, prolonged fluorescence stability
indicated a significant resistance to irreversible photo-
bleaching. The wonderful multilabeling properties make
1 highly suitable for the mapping of Hg2þ distribution in
living cellꢀEPSꢀmineral aggregates, which may provide
breakthrough insight into the correlation between Hg2þ
and organic substances such as cells, microbes, and bio-
films. To the best of our knowledge, this is the first six-
membered spirocycle fluorescent probe which displays
such promising properties for Hg2þ detection.
As expected, probe 1 is colorless and emits no fluores-
cence in ethanolꢀPBS (4/6, v/v, pH 7.4) solution. Notably,
upon addition of Hg2þ, an intense absorption band cen-
tered at 556 nm (Figure 2a), coupled with a brilliant pink
coloration. Concomitantly, a strong orange emission band
appeared around 574 nm (Figure 2b) with a high fluo-
rescent quantum yield (Φ = 0.87). Fluorescent titration
with 20 μM of 1 demonstrated that the detection limit was
Figure 3. Absorption (a) and fluorescence intensity (b) changes
of 1 (20 μM) upon the addition of various metal ions (20 μM) in
ethanolꢀPBS (4/6, v/v, pH 7.4) solution. Red bars represent the
fluorescence response of 1 to the metal ions of interest. 1, blank;
2, Liþ; 3, Naþ; 4, Kþ; 5, Agþ; 6, Ba2þ; 7, Ca2þ; 8, Mg2þ; 9, Cd2þ
;
10, Mn2þ; 11, Co2þ; 12, Fe2þ; 13, Ni2þ; 14, Zn2þ; 15, Pb2þ; 16,
Cu2þ; 17, Fe3þ; 18, Cr3þ; 19,Al3þ; 20, Hg2þ. Black bars repre-
sent the subsequent addition of Hg2þ (20 μM) to the above
solutions (excitation at 550 nm; emission at 574 nm).
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Microcalorimetry was used for a better understanding
of the mechanism.10 The enthalpy at 298.15 K was mea-
sured to be ꢀ24.09 ( 0.02 kJ/mol, revealing that the
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Org. Lett., Vol. 15, No. 17, 2013
4335

ring-opening process is exothermic and spontaneous. Job’s
plot showed a 1:1 stoichiometry between 1 and Hg2þ with
an association constant of 1.02 ꢁ 106 Mꢀ1, indicating the
strong binding affinity of 1 to Hg2þ (Figure S5). The mass
spectrum manifested a peak at m/z 826.1943, assigned as
[1 þ Hg2þ þ Clꢀ]þ, providing further evidence for the
binding mode. The absorption at 1678 cmꢀ1 in IR analysis
did not shift to lower frequency upon addition of Hg2þ
(Figure S10), indicating the carbonyl moiety was not in-
volved in the coordination. Delightfully, a single crystal of
the possible intermediate was obtained, and it suggested
thatthe coordination of the Hg2þ selectively occurred at the
N atoms of the hydrazine unit and benzothiazole moiety.
To better understand the binding between Hg2þ and 1,
DFT11-NBO analysis12 focusing on the electron lone pairs
(LP) was performed for 1.12 The LP of the N atoms of the
hydrazine and benzothiazole moieties were computed to be
the most possible ones to interact with Hg2þ (Figure S16).
Based on these results, the most possible structure of the
complex [1 þ Hg2þ þ Clꢀ]þ was proposed and validated by
its lower energy compared to other possible structures
(Figure S17). It can be concluded that the coordination
of Hg2þ to the N atoms of the hydrazine and benzothiazole
moieties reasonably promoted the opening of the spirocycle
thus inducing the color and fluorescent changes (Figure 4).
addition of Hg2þ. Both the absorption and emission
corresponded mainly to the orbital transition between
HOMO and LUMO (Figure 5). These frontier MOs were
almost entirely distributed within the xanthene moiety
absolutely and thus the corresponding dipole integral
would be large. Finally the transition dipole moment
between the S0 and S1 states was large as well as the
strengths of the corresponding absorption and emission.
Although the calculations underestimate the absorption
wavelength by ∼70 nm, the theoretical emission wave-
length 543 nm was quite close to the experimental value
(within ∼30 nm). As shown in Figure 5, the Stokes shift
mainly arose from the lower LUMO energy (ꢀ3.17 ev)
induced by the geometry relaxation at the potential energy
surface of the S1 state.
The fluorescent response changed inappreciably in the
pH range of 4ꢀ8 (Figure S6) and had little interference
from sulfur compounds (Figure S7). This is highly desir-
able for quantifying Hg2þ in biological systems. As a
proof-of-concept, HeLa cells were pretreated with 20 μM
of 1 or vehicle control for 4 h at 37 °C and then incubated
with 20 μM HgCl2 in the same culture medium. The
preliminary experiments in HeLa cells demonstrated that
1 is especially suited for in vivo imaging (Figure S8).
Figure 4. Proposed recognition process for the fluorescent
changes upon addition of Hg2þ
.
The absorption and fluorescent emission of the complex
were theoretically modeled at the TDDFT13 level based on
the optimized structure of the ground S0 state and the first
excited S1 state. The theoretical result was consistent with
the experimental observation of strong fluorescence upon
Figure 5. Theoretical wavelength (λ), excitation energy, oscillator
strength (f), relevant frontier MOs (3D distribution and orbital
energy), and corresponding CI coefficient of the absorption and
emission of the complex [1 þ Hg2þ þ Clꢀ]þ in ethanol solvent.
Probe 1 was then used to map the sorption of Hg2þ to
bacteriaꢀEPSꢀmineral aggregates under anoxic conditions14
(Figure S8). Reflection images of bacteriaꢀEPSꢀmineral
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Org. Lett., Vol. 15, No. 17, 2013

advantage of analyzing the samples in an close to natural,
hydrated state and thus avoids artifacts from sample
preparation such as relocation of metals and dissolution/
removal of EPS due to washing steps as they are
required for electron microscopy sample preparation.
Moreover, the approach could be a powerful tool
to selectively screen bacterial strain and microbial
biofilms for efficient removal of mercury from con-
taminated environments.
In summary, we developed a rhodamine derivative 1
witha unique six-membered spirocycle asa novel probe for
Hg2þ. The extension of the spirocycle from a five- to a
stretched nonplanar six-member ring greatly improved the
properties of 1, especially in terms of selectivity, sensitivity,
and photostability. The recognition behaviors are investi-
gated both experimentally and computationally. Introduc-
tion of the benzothiazole moiety has been proven to
facilitate the formation of the six-membered spirocycle
and the selective recognition of Hg2þ. Confocal laser
scanning microscopy results show the applicability of 1
for multilabeling approaches. This should contribute to a
significant breakthrough in understanding the correlation
between Hg2þ and related cells and organic substances.
More importantly, this probe is also a promising candidate
for super-resolution fluorescence imaging with a lateral
resolution of ∼20 nm using direct stochastic optical re-
construction microscopy.16
Figure 6. Single cell scale map the sorption of mercury to
bacteriaꢀEPSꢀmineral aggregates incubated with 20 μM
Hg2þ. Simultaneously incubated sample with 20 μM probe 1
(b), fluorescent nucleic acid stain (a), and lectin conjugate (c) for
3 h at 20 °C (λex = 488, 561, 635 nm). The overlay image of (a),
(b), and (c) is shown in (d).
aggregates showed the morphological diversity. The over-
lay of fluorescence and reflection images revealed that
Hg2þ was mainly adsorbed on the cells’ surfaces. To
better understand the mechanism and site of binding,
DNA- and polysaccharide-specific fluorescent dyes15
were applied simultaneously with 1 (Figure 6). On a single
cell level, we show that capsule-like EPS structures en-
close the SW2 cell. The structures were collocated with the
fluorescence signal of 1 which indicated the Hg2þ adsorp-
tion to the EPS. The overlay image indicated that high
concentrations of Hg2þ were located in the capsule-like
EPS structure. This seemed to provide binding sites for
Hg2þ and thus might protect the cell from mercury
toxicity. Our results indicated that 1 could be used
to map Hg2þ distribution in living cellꢀEPSꢀmineral
aggregates under anoxic conditions. In comparison to
other analytical approaches, fluorescence labeling has the
Acknowledgment. We are grateful for the support from
the NSF of China (No. 21272184, 21103137, 20972124),
Special Science Research Foundation of Education Com-
mittee in Shaanxi Province (No. 12JK0584), Shaanxi
Science and Technology Coordination Innovation Engi-
neering Project (No. 2011K12-77), and the Emmy-Noether
fellowship program of the DFG to M.O. (OB 362/1-1).
Yin, B. wants to express his thanks to Prof. Yuanhe Huang
(College of Chemistry, Beijing Normal University) for his
great help.
Supporting Information Available. Experimental de-
tails, optical spectra, IR, NMR, MS spectra, and compu-
tational details. This material is available free of charge
via the Internet at http://pubs.acs.org.
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B.; Volke, F.; Dynes, J. J.; Hitchcock, A. P.; Lawrence, J. R. FEMS
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The authors declare no competing financial interest.
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