HgII-selective fluorescent indicator: One-step synthesis

Article abstract of DOI:10.1002/ejoc.201200126

Fluorescent indicator 2 was synthesized by a single-step reaction. The indicator underwent an Hg^II-selective reaction (hydrolysis/ decarboxylation sequence) in the presence of Hg^II, which was verified by using LC-MS and NMR techniques.

Full text of DOI:10.1002/ejoc.201200126

SHORT COMMUNICATION
DOI: 10.1002/ejoc.201200126
HgII-Selective Fluorescent Indicator: One-Step Synthesis
Seul-Bi Kim[a] and Dong-Gyu Cho*[a]
Keywords: Mercury / Indicators / Fluorescence / Sensors / Reaction selectivity
Fluorescent indicator 2 was synthesized by a single-step re-
action. The indicator underwent an HgII-selective reaction
(hydrolysis/decarboxylation sequence) in the presence of
HgII, which was verified by using LC-MS and NMR tech-
niques.
thiocarbonyl transformation. On the other hand, the detec-
tion limits are relatively high in such probes (0.89 μm),[8b]
and reactions are also slow in the case of naphthoflavo-
thiones.[8a] Other strategies show better sensitivity, but they
require multiple-step syntheses. Thus, we became interested
in finding an HgII-selective reaction that can be easily in-
stalled. Finding such a reaction allows us to report an HgII-
selective indicator synthesized in a single step from a com-
mon starting material. The indicator undergoes HgII-selec-
tive hydrolysis of thiol followed by decarboxylation (hydrol-
ysis/decarboxylation sequence) in the presence of HgII.
Introduction
Many metal ions play important roles in ecology and
biology. Nevertheless, one heavy metal ion, the HgII cation,
is extremely toxic with no benign effect. Mercury cations
coordinate easily to biological ligands, such as proteins,
DNA and enzymes, which causes significant damage to the
brain, kidney, stomach and central nervous system.[1]
Therefore, many different kinds of chemical sensors have
been developed as prototype tools for detecting toxic HgII
ions in environmental and biological samples. Recently, an-
alyte-selective reaction indicators[2] have attracted consider-
able attention since Czarnik’s seminal work[3] owing to their
high sensitivity and selectivity compared to traditional re-
versible indicators.[4] This approach relies on an irreversible
chemical reaction rather than reversible interactions. Con-
sequently, the sensitivity and selectivity of the reaction indi-
cators depend on an HgII-selective reaction instead of a
shape-recognition process (lock-and-key concept). This ap-
proach allows the chemical structures of sensors to be much
simpler than traditional reversible indicators, because they
require only reaction and signaling units. Thus far, two
types of HgII-selective reaction-based detection strategies
have been developed. Recently, HgII-selective hydration re-
actions of alkynes or alkenes were used to detect HgII.[5]
The large body of HgII-selective indicators takes advantage
of the thiophilic nature of HgII. This thiophilicity of HgII
is involved in many different HgII-selective reactions, such
as ring opening of spirocyclic systems,[6] intramolecular cy-
clic guanylation of thiourea derivatives,[7] desulfurization of
thiocarbonyl compounds,[3,8] desulfurization/lactonization
reaction[9] and deprotection of dithiol-protected alde-
hydes.[10] In particular, a desulfurization strategy is attract-
ive, because it is easily installed by a simple carbonyl-to-
Results and Discussion
The thiocarbamate group (reaction unit) has been used
for protecting thiol groups rather than amine groups with
ethyl isocyanate or its derivatives in a few cases.[11] To install
the reaction unit and minimize synthetic efforts, a commer-
cial fluorescent dye that contains an amine group was cho-
sen. 7-Amino-4-methyl-2H-chromen-2-one (1) was con-
verted into its isocyanate by triphosgene and then treated
in situ with ethanethiol (Scheme 1). This method afforded
HgII-selective indicator 2 in 73% yield. The HgII-selective
hydrolysis/decarboxylation sequence of compound 2 was
monitored by UV/Vis spectroscopy, as shown in Figure 1a.
In the absence of HgII, the absorption maximum of com-
pound 2 was observed at 329 nm. After the addition of
2 equiv. of HgCl2, all spectral changes were complete within
5 min. Encouraged by these results, an effort was made to
increase the response. The same reaction was carried out by
using a 3.3 μm solution of compound 2 in DMSO/water
(1:9, v/v) (10 mm PBS buffer, pH = 7.0) and monitored by
fluorescence spectroscopy. Upon the addition of various
quantities of HgII to the solutions, a significant decrease
in fluorescence intensity at 403 nm and an increase in the
fluorescence emission band centered at 442 nm were ob-
served (Figure 1b). The fluorescence intensity at 442 (or
I442/I403) increased linearly with increasing HgII concentra-
tion (0.33–0.2 μm) (Figure S1, Supporting Information).
[a] Department of Chemistry, Inha University,
253 Yonghyundong, Namgu, Incheon 402-751,
Republic of Korea
Fax: +82-32-867-5604
E-mail: dgcho@inha.ac.kr
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201200126.
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S.-B. Kim, D.-G. Cho
SHORT COMMUNICATION
Further dilution studies of compound 2 (0.33 μm) upon the observed in the presence of 2 equiv. of HgII under a labora-
addition of HgII were also carried out. The reactions were tory UV light (365 nm), as shown in Figure 2b. In contrast,
slow under these conditions (Figure S2). Therefore, the re- there was no change in fluorescence color in the presence
actions were carried out for 1 h, and the fluorescence inten- of other metal ions.
sity at 442 nm was observed to increase linearly with in-
creasing HgII concentration (33–330 nm), as shown in Fig-
ure S3. The corresponding detection limit was determined
to be less than 33 nm.
Scheme 1. Synthesis of the HgII indicator 2.
Figure 2. (a) Fluorescence responses of 2 (3.3 μm) with various
metal ions (2 equiv.) in DMSO/water (1:9, v/v) (10 mm PBS buffer,
pH = 7.0) (λex = 342 nm). All measurements were conducted
10 min after the addition of various ions. (b) Fluorescence change
of 2. Left to right: CdII, FeIII, CoII, NiII, MgII, HgII, 2 only, CuII,
and AuIII. (c) Metal-ion selectivity of 2 (3.3 μm). The red bars rep-
resent the emission intensity of 2 in the presence of other cations
(2 equiv.). The blue bars represent the emission intensity that oc-
curs upon the subsequent addition of 2 equiv. of HgII to the above
solution.
Figure 1. (a) UV/Vis spectra of indicator 2 (33 μm) in DMSO/water
Competition experiments were also carried out by add-
(1:9, v/v) (10 mm PBS buffer, pH = 7.0) were recorded before and
ing 2 equiv. of HgII to buffered solutions of 2 equiv. of other
immediately after the addition of HgCl2 (2 equiv., 6.60ϫ10–3 m in
metal ions, as shown in Figure 2c. All competitive metal
ions did not interfere with the detection of HgII ions, which
indicates that the system of compound 2/HgII was barely
affected by these coexistent ions (Figure 2c).
water) at 25 °C. (b) Fluorescence responses were recorded 10 min
after adding various quantities of HgII (0–1.2 equiv. of HgII) to
compound 2 (3.3 μm) in DMSO/water (1:9, v/v) (10 mm PBS buffer,
pH = 7.0).
Rational analyses of the HgII-selective reaction were per-
To validate the selectivity of compound 2 in practice, formed by using liquid chromatography-mass spectrometry
2 equiv. of each cation (CuI, CuII, AuIII, CoII, HgII, FeIII (LC-MS). The addition of HgII to a 2 mL sample of indi-
ZnII, AgI, PbII, NaI, PtII, FeII, NiII, AlIII, MgII, KI, CdII, cator 2 resulted in the formation of a new peak in LC-MS
and CaII) were added to the solutions (3.3 μm) in DMSO/ that was consistent with the mass of compound 1 shown in
water (1:9, v/v) (10 mm PBS buffer, pH = 7.0), and the fluo- Figure 3. The relative small intensity of compound 2 results
rescence spectra were recorded 10 min later. As shown in from the relative intensities of compounds 1 and 2 at
Figure 2a, only HgII caused a change in the fluorescence of 254 nm (cf. Figure 2a and Figure 3). The formation of com-
compound 2. A blue to cyan fluorescence color change was pound 1 is presumably preceded by hydrolysis followed by
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HgII-Selective Fluorescent Indicator: One Step Synthesis
Figure 3. Peaks in the liquid chromatograms that undergo a change upon the addition of HgII (2 equiv.) to 2 mL of a solution of indicator
2 [3.3 μm in CH3CN/water (2:8, v/v) (10 mm PBS buffer, pH = 7)] (left) and their corresponding mass spectra as determined by LC-MS
(right).
Scheme 2. Proposed mechanism of the HgII-selective indicator reaction.
a decarboxylation reaction (Scheme 2). It is also important
Experimental Section
to note that the nitrogen atom of the thiocarbamate group
Ethyl (S)-4-Methyl-2-oxo-2H-chromen-7-ylcarbamothioate (2): To a
is critical to the reaction. The scope and details of the reac-
mixture of 7-amino-4-methylcoumarin (50 mg, 0.29 mmol) and
tion will be published elsewhere. NMR spectroscopy also
TEA (0.4 mL, 2.87 mmol) in CH2Cl2 (5 mL) cooled in an ice bath
provided mechanistic support for this reaction. Upon the
addition of HgII to a solution of compound 2 in [D6]-
DMSO/D2O (8:2, v/v), we observed only signals of 2 and 1
in the time-dependent 1H NMR spectra during the reaction
(Figure S4). The reaction was complete within 30 min.
Nonetheless, the present HgII-promoted reaction proceeded
rapidly with higher water contents, even at the μm level
(Figures 1 and 2). The changed chemical shifts of com-
pound 2 were superimposed on the original spectrum of
compound 1 (Figure S4). In addition, the reaction requires
a 2:1 stoichiometry (indicator/HgII), which was confirmed
by a UV/Vis spectroscopic titration (Figure S5).
was added a solution of triphosgene (126 mg, 0.43 mmol) in THF
(2 mL) dropwise for 30 min. After 1 h, ethanethiol (43.6 ul,
0.57 mmol) was added to the resulting solution still cooled in an
ice bath, and stirring was continued for 3 h. The reaction mixture
was diluted with CH2Cl2 and then washed with water. The organic
layer was separated, dried with Na2SO4, and subjected to filtration.
After the organic solvent had been removed under reduced pres-
sure, the resulting residue was purified by column chromatography
on silica gel (EtOAc/hexane, 1:1) to afford indicator 2 as a white
solid (55 mg, 0.21 mmol, 73% yield). 1H NMR (400 MHz, [D6]-
DMSO): δ = 1.22 (t, J = 7.4 Hz, 3 H), 2.35 (d, J = 1.2 Hz, 3 H),
2.86 (q, J = 7.4 Hz, 2 H), 6.21 (d, J = 1.2 Hz, 1 H), 7.38 (dd, J =
8.7, 2.1 Hz, 1 H), 7.55 (d, J = 2.3 Hz, 1 H), 7.68 (d, J = 8.7 Hz, 1
H) ppm. 13C NMR (100 MHz, [D6]DMSO): δ = 15.6, 18.0, 23.5,
105.3, 122.3, 114.8, 115.0, 126.1, 142.1, 153.0, 153.7, 160.0,
165.8 ppm. HRMS (CI): calcd. for C13H14NO3NS [M + H]+
264.0694; found 264.0696.
Conclusions
A simple fluorescent indicator that undergoes a new type
of reaction (hydrolysis/decarboxylation sequence) only in
the presence of HgII was developed. The HgII-detection
mechanism was verified by using LC-MS and NMR tech-
niques. In addition, indicator 2 required only a single-step
synthesis. The properties of HgII indicator 2 show the essen-
tial qualities of reaction-based indicators. Overall, this sim-
ple compound synthesized in a single step can achieve
higher selectivity and sensitivity toward HgII over other
metal ions.
Supporting Information (see footnote on the first page of this arti-
cle): Experimental procedures, spectroscopic titrations, and spec-
troscopic data for fluorescent indicator 2.
Acknowledgments
This research was supported by an Inha University Research Grant
and the Basic Science Research Program through the National Re-
search Foundation of Korea (NRF) funded by the Ministry of Edu-
cation, Science and Technology (grant no. 2010-0004206).
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Received: February 3, 2012
Published Online: ᭿
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