Selective ratiometric detection of Hg2+ in pure water using a phenoxazinium-based probe

Article abstract of DOI:10.1016/j.tetlet.2011.03.013

A ratiometric, near-infrared, and fully water-soluble probe, a phenoxazinium-based chemosensor bearing an anilino thiaazacrown, was successfully synthesized and characterized. The use of this probe for the selective ratiometric detection of Hg²⁺

Full text of DOI:10.1016/j.tetlet.2011.03.013

Tetrahedron Letters 52 (2011) 2492–2495
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Selective ratiometric detection of Hg2+ in pure water using
a phenoxazinium-based probe
⇑
⇑
Xue-Bo Yang, Jian-Feng Ge , Qing-Feng Xu, Xiao-Ling Zhu, Na-Jun Li, Hong-wei Gu, Jian-Mei Lu
Key Laboratory of Organic Synthesis of Jiangsu Province, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou 215123, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A ratiometric, near-infrared, and fully water-soluble probe, a phenoxazinium-based chemosensor bearing
an anilino thiaazacrown, was successfully synthesized and characterized. The use of this probe for the
selective ratiometric detection of Hg2+ in pure water is reported. The probe shows good selectivity for
Hg2+, and a large blue shift (75 nm) of the complex’s absorption maximum was observed.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 19 November 2010
Revised 24 December 2010
Accepted 3 March 2011
Available online 8 March 2011
Keywords:
Hg2+ detection
Ratiometric
Water-solution
Phenoxazinium
The mercury(II) ion is considered to be one of the most toxic
metal cations in the environment and is known to seriously affect
human health.1 Sensitive organic ligands for the detection of Hg2+
,
which can provide immediate optical feedback and which do not
require sophisticated instrumentation and sample preparations,
are highly valued. Many detection methods for Hg2+ based on or-
ganic small-molecules have been examined.2 Current research
shows that: (1) Ratiometric monitoring3 is a superior design for a
probe because it can increase the selectivity and sensitivity of
the measurement and eliminate most or all of the interfering fac-
tors by self-calibrating the two emission or absorption bands.4
(2) It is very important that the probe has absorption or emission
bands in the longer wavelengths.2b,3g,5 Light in the near-infrared
(NIR) region (650–900 nm)6 causes minimal photo damage7 and
can avoid the influence of autofluorescence.8 (3) A fully water-
soluble probe is an important goal, as it facilitates a probe’s
application in environmental or biological fields of study.9
However, few aqueous probes3a,b,10 have been reported when
compared to the large number of organic2a or water-compati-
bile2b–d probes. In light of these precedents, we committed our-
selves to the development of a new water-soluble ratiometric
sensor with long-range excitation and emission as well as pros-
pects for environmental or biological utility.
The symmetric and unsymmetrical 3,7-bis(dialkylamino)-phen-
oxazin-5-ium derivatives, which contain a phenoxazinium cation
with absorbs in the 640–650 nm region and have corresponding
Scheme 1. Synthesis of phenoxazinium 1a.
emission bands between 650 and 670 nm,11 have comparatively
low toxicity according to previous in vitro and in vivo assay
results.12a,b We assume that 3,7-bis(dialkylamino)phenoxazin-5-
ium derivatives that are appended with a 1,4-dioxa-7,13-dithia-
10-azacyclopentadecane unit (e.g., 1a) coordinate with Hg2+
through the anilino thiaazacrown.2b,3a,d The absorption and emis-
⇑
Corresponding authors. Address: 199 Ren’ai Road, Suzhou Industrial Park,
Suzhou 215123, China (J.-M.L.). Tel.: +86 512 6588 0368; fax: +86 512 6588 0367.
sion properties are modulated by the extended p–
p conjugation
between the nitrogen atom and phenoxazinium.11
E-mail addresses: gejianfeng@suda.edu.cn (J.-F. Ge), lujm@suda.edu.cn (J.-M. Lu).
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.03.013

X.-B. Yang et al. / Tetrahedron Letters 52 (2011) 2492–2495
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Figure 1. (a) Changes in the UV–vis spectra of 1a (50 Â 10À6 M in water) upon titration with Hg(AcO)2 from 50 Â 10À6 M to 300 Â 10À6 M. Inset: color change of 1a in the
visible region. (i) 1a solution in water; (ii) (i) + 6 equiv Hg2+. (b) Changes in the UV–vis spectra of 1a (40 Â 10À6 M in water) upon addition of 5 equiv mercury ion and
subsequently an excess of EDTA. Inset: color change of 1a in the visible region. (i) 1a solution in water; (ii) (i) + 5 equiv.Hg2+; (iii) (ii) + excess of EDTA. (c) Job plot analysis of
1a and Hg2+ in water, the total molar concentration of 1a and Hg2+ is 60 Â 10À6 M.
3-(Diethylamino)-7-(1,4-dioxa-7,13-dithia-10-azacyclo
pen-
useful probes to detect the influence of the Hg2+ complexation.
Addition of Hg2+ to 1a induces a large downfield shift of the 8-H
and 6-H signals, which also clearly indicates a decrease in the elec-
tron donating ability of the amino group.
tadecan-10-yl) phenoxazin-5-ium chloride (1a) was obtained in
30% yield by reacting 1,4-dioxa-7,13-dithia-10-(3-methoxy-4-
nitrosophenyl)-10-azacyclopentadecane (4) with 3-diethyl-amino-
phenol (Scheme 1). Compound 1a exhibits good solubility in water
and polar solvents, such as methanol and chloroform.
Control experiments were performed to evaluate the selectivity
of probe 1a toward Hg2+. The influence of the addition of Hg2+, Na+,
Mg2+, Ca2+, K+, Cd2+, Zn2+, Ni2+, Cr3+, Cu2+, Pb2+, Mn2+, Fe3+, or Ag+ on
the absorption properties of 1a was studied (Fig. 3a). The concen-
Figure 1a shows the changes in the absorption spectrum of 1a
as a function of Hg2+ concentration in water at room temperature.
The UV–vis spectrum of 1a in water is characterized by a very in-
tration of the metal ions was 0.1 Â 10À3 M, except for K+, Na+, Mg2+
,
tense band centered at 653 nm (
e
= 76000 MÀ1 cmÀ1) that is
and Ca2+ (100Â10À3 M) because the latter would likely be present
in higher concentrations in real samples. It should be noted that
the addition of Hg2+ leads to a significant change of the absorption
of 1a, a blue-shift in the absorption maximum of 75 nm was ob-
served, and the solution color changed from light blue to dark blue
(Fig. 1a). A slight decrease in the absorption intensity was detected
upon addition of Ag+ (Fig. 3a); and the competition experiments
show that it has no significant influence when the samples con-
tained different metal cations (Fig. S2a, Supplementary data).
The absorption intensity ratio I530/I653 (Fig. 3b) of the UV–vis
spectrum shows that the shift of the absorption maximum of 1a
upon complexation with Hg2+ allows the analysis of the UV–vis
titration by a ratiometric plot, that is, a plot of I530/I653 versus
Hg2+ concentration. In contrast, there is almost no change in the
responsible for the light blue color of the solution. During photo-
metric titrations of 1a with Hg2+, a blue shift of the long-wave-
length absorption maximum from 653 nm to 578 nm
(e
= 34000 MÀ1 cmÀ1) (with one isosbestic point at 590 nm) was
observed, which may be the result of the complexation of Hg2+
with the crown ether unit. Such a large blue-shift (75 nm) makes
the color of the solution change from light blue to dark blue
(Fig. 1a). The subsequent addition of an excess of ethylenediamine
tetra-acetic acid (EDTA) resulted in recovery of the original color
(Fig. 1b).
According to the linear Benesi–Hildebrand expression2a, the
measured absorbance [1/(A-A0)] at 650 nm varied as a function
of 1/[Hg2+] in a linear relationship indicating the 1:1 stoichiometry
between the Hg2+ ion and 1a (Fig. S4b, Supplementary data). On
the basis of 1:1 stoichiometry and UV–vis titration data in Fig-
ure 1a, the association constant of 1a with Hg2+ ion in water was
found to be 4.5 Â 103 MÀ1 (Fig. S4b, Supplementary data). Job plot
analysis2e of the UV–vis titrations carried out in water revealed a
maximum at a 50% mole fraction, in accord with the proposed
1:1 binding stoichiometry(Fig. 1c).
I530/I653 ratio (0.18) upon addition of Cd2+, Zn2+, Ni2+, Cr3+, Cu2+
,
Pb2+, Co2+, Fe3+, Na+, Mg2+, or Ca2+. However, the addition of Ag+ in-
duced a slight increase of the I530/I653 value (0.39). At the employed
concentration of the probe (50 Â 10À6 M), the limit of detection for
Hg2+ is 10À6 M, and the linear range of the detection is 20–
120 Â 10À6 M (Fig. S1a and S1b Supplementary data).
3,7-Di(piperidinyl)phenoxazin-5-ium chloride(1b, Scheme 2)
was used to practice the same procedure, and there was not any
difference in the absorption spectra(Fig. S3a, Supplementary data).
This indicated that the absorption is changed not by possible com-
bination13 between the phenoxazinium skeleton and Hg2+ but by
the chelation between the anilino thiaazacrown and Hg2+. Regard-
ing possible applications of ligand 1a, these conditions permit the
detection and quantification of Hg2+ in industrial waste water.
Note that fluorometric detection of Hg2+ is also possible with
compound 1a. The addition of Hg2+ causes a decrease and a
blue-shift in the fluorescence of compound 1a. As there is slight
decrease of the fluorescence intensity of 1b and Hg2+ (Fig. S3b,
Supplementary data), we assume that the emission is mainly chan-
ged by the combination between the anilino thiaazacrown with
Hg2+, and it is also slightly influenced by the chelation between
The absorption spectra of 1aÁHg2+ clearly indicate that the
absorption band at 653 nm has shifted and developed a new band
at 578 nm. The blue-shifted absorption indicates that the complex
formed between 1a and Hg2+ alters the properties of the amino
functionality, significantly suppressing its ability to donate elec-
tron density to the phenoxazinium chromophore.
The complexation of Hg2+ with compound 1a was demonstrated
by proton nuclear magnetic resonance spectra (1HNMR) spectros-
copy (Fig. 2). Thus, upon addition of Hg2+, the aliphatic protons
of the methylene units next to the sulfur atoms, H-b and H-c, expe-
rience large downfield shifts of 0.55 ppm and 0.46 ppm, respec-
tively, whereas the methylene protons adjacent to the oxygen
atoms, H-d and H-e, show slight downfield shifts (0.09 ppm). The
chemical shifts of the aromatic protons concurrently move to low-
er field (0.09–0.28 ppm), indicating the reduced donor strength of
the amino substituent as a result of the complexation of 1a with
Hg2+. The peaks corresponding to the protons at C-8 and C-6, which
are located at the b-position of the amino group, are particularly
the phenoxazinium skeleton and Hg2+
.
To determine the amount of Hg2+ required to induce fluores-
cence quenching of 1a, titration experiments were performed
(Fig. 4); when 6 equiv of Hg2+ was added, the emission of 1a at

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X.-B. Yang et al. / Tetrahedron Letters 52 (2011) 2492–2495
Figure 2. Top: 1H NMR spectra of free 1a in CD3OD. Bottom: 1H NMR spectra of 1a in the presence of Hg2+ (2 equiv) in a mixed solvent system of D2O and CD3OD (D2O:
CD3OD = 1:5).
Figure 4. Emission spectra of compound 1a in the presence of increasing Hg2+
concentration (from 50 Â 10À6
M
to 300 Â 10À6 M) in water. The excitation
wavelength was 590 nm with a 5 nm slit width. The concentration of 1a was
50 Â 10À6 M.
695 nm was quenched and blue-shifted. The detection limit of the
sensor at this concentration for Hg2+ is about 50 Â 10À6 M (Fig. S1c,
Supplementary data). The control experiments and competition
experiments were also performed to evaluate the selectivity of
probe 1a toward Hg2+ upon fluorescence spectra. The experiments
indicate the probe 1a has high selectivity toward Hg2+ using fluo-
rescence spectra (Fig. S2b and S2c Supplementary data).
The emission of Hg2+ complexed 1a is quenched at low pH
(Fig. S4a, Supplementary data), presumably owing to protonation
of the complex in the exited state because neither the absorption
of 1a in the absence or the presence of Hg2+ (0.1 Â 10À3 M), nor
the emission of uncomplexed 1a change with decreasing pH.3a
These results demonstrate the pH dependence of the fluorometric
performance of 3,7-bis(dialkylamino) phenoxazin-5-ium conju-
gates. Therefore, if the pH of the medium is known, compound
1a is still an opportune Hg2+ sensitive probe as demonstrated by
calibration curves determined at given pH values.3a
Figure 3. (a) Changes in the UV–vis spectra of 1a (50 Â 10À6 M in water) upon
addition of different metal cations (0.1 Â 10À3 M: Hg2+, Cd2+, Zn2+, Ni2+, Cr3+, Cu2+
,
Pb2+, Fe3+, Ag+, Mn2+; 100 Â 10À3 M: K+, Na+, Mg2+, Ca2+). (b) Relative absorption
intensities of the ligand 1a (c = 50 Â 10À6 M), given as the ratio I530/I653 upon
addition of different metal cations.
In conclusion, we present an example of the ion probe’s selec-
tion for the suitable chromophore or fluorophore from the existing
or potential drug molecules. A phenoxazinium-based chemosensor
bearing an anilino thiaazacrown (1a) as a promising analytical tool
for UV–vis and fluorometric mercury detection with long-range
excitation and emission. Specifically, this probe is one of the few
Scheme 2. Structure of phenoxazinium 1b.

X.-B. Yang et al. / Tetrahedron Letters 52 (2011) 2492–2495
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Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2011.03.013.
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