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).
(7) (a) Ando, S.; Koide, K. J. Am. Chem. Soc. 2011, 133, 2556. (b)
Yang, M.; Thirupathi, P.; Lee, K. Org. Lett. 2011, 13, 5028. (c)
Vedamalai, M.; Wu, S. P. Org. Biomol. Chem. 2012, 10, 5410.
(8) Wu, C.; Bian, Q. N.; Zhang, B. G.; Cai, X.; Zhang, S. D.; Zheng,
H.; Yang, S. H.; Jiang, Y. B. Org. Lett. 2012, 14, 4198.
(9) (a) Wang, J. B.; Qian, X. H. Chem. Commun. 2006, 109. (b) Li, D.;
Wieckowska, A.; Willner, I. Angew. Chem., Int. Ed. 2008, 47, 3927. (c)
Shiraishi, Y.; Sumiya, S.; Kohno, Y.; Hirai, T. J. Org. Chem. 2008, 73,
8571. (d) Kumar, M.; Kumar, N.; Bhalla, V.; Singh, H.; Sharma, P. R.;
Kuar, T. Org. Lett. 2011, 13, 1422.
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
(10) (a) Cardona-Martinez, N.; Dumesic, J. A. Adv. Catal. 1992, 38,
149. (b) Gaisford, S. Adv. Drug Delivery Rev. 2012, 64, 431.
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