Hg2+-selective dual signaling probe based on a thio-functionalized rhodamine B hydroxamic acid

Article abstract of DOI:10.1016/j.tetlet.2015.06.085

Abstract A new Hg²⁺-selective signaling system based on a thio-functionalized rhodamine B hydroxamic acid was investigated. A thio-analogue of rhodamine B hydroxamic acid had a distinct Hg²⁺-selective signaling behavior in aqueous solution, whereas its parent hydroxamic acid was completely unreactive toward any metal ions. Selective chromogenic and fluorogenic signaling of Hg²⁺ by the thio-functionalized rhodamine B hydroxamic acid in aqueous solution was possible with a detection limit of 1.0 × 10^-6 M. Hg²⁺ determination based on paper test strips was conducted as a practical application.

Full text of DOI:10.1016/j.tetlet.2015.06.085

Tetrahedron Letters 56 (2015) 4919–4922
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Hg2+-selective dual signaling probe based on a thio-functionalized
rhodamine B hydroxamic acid
⇑
Yong Ae Jeong, Jiyoung Choi, Suk-Kyu Chang
Department of Chemistry, Chung-Ang University, Seoul 156-756, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
A new Hg2+-selective signaling system based on a thio-functionalized rhodamine B hydroxamic acid was
investigated. A thio-analogue of rhodamine B hydroxamic acid had a distinct Hg2+-selective signaling
behavior in aqueous solution, whereas its parent hydroxamic acid was completely unreactive toward
any metal ions. Selective chromogenic and fluorogenic signaling of Hg2+ by the thio-functionalized rho-
damine B hydroxamic acid in aqueous solution was possible with a detection limit of 1.0 Â 10À6 M. Hg2+
determination based on paper test strips was conducted as a practical application.
Article history:
Received 27 May 2015
Revised 20 June 2015
Accepted 26 June 2015
Available online 30 June 2015
Keywords:
Ó 2015 Elsevier Ltd. All rights reserved.
Hg2+ signaling
Chromogenic probe
Fluorogenic probe
Rhodamine hydroxamic acid
Thionation
The development of smart sensors or probes for the selective
sensing and visualization of important metal ions has attracted
much research interest,1 and many elaborate chemosensors based
on fluorescein, rhodamine, and other functional dyes are con-
stantly being designed for this purpose. Among the various molec-
ular platforms used for the design of sensing materials, rhodamine
fluorophores are particularly widely used because of their favor-
able optical properties.2 In particular, hydroxamic acid derivatives
of rhodamine have recently attracted much research interest.
Rhodamine hydroxamic acids and their derivatives have exhibited
calix[4]arene-based amide derivatives was used to target thiophilic
metal ions, such as Hg2+ and Cd2+ 12
Simple rhodamine fluo-
.
rophores have been transformed into thiolactones by thionation
to change them into Hg2+-selective sensors.13 Switching the recog-
nition preference of rhodamine B spirolactam by replacing one
atom resulted in the design of rhodamine B thiohydrazide for the
recognition of Hg2+ in aqueous solution.14
However, the thio-analogues of many dyes have been used as
chemodosimeters for the signaling of many important species,
such as Hg2+, Cu2+, and various oxidants. In particular, thio-func-
tionalized chromophores and fluorophores are the basis of the
design of desulfurization-based chemodosimeters, which exploit
the thiophilicity of Hg2+ and its critical role in desulfurization reac-
tions.15 Since the development of Czarnik’s anthracene-based
thioamide,11a many probes have been developed using similar
Hg2+-induced desulfurizations of thiocarbonyl functional groups,
such as thiocoumarins,16 thioureas,17 and 8-hydroxyquinoline-
based thioamide derivatives.18 In addition, many chemodosimeters
based on the sequential reactions of Hg2+-induced desulfurization
followed by cyclization have also been reported.19
interesting signaling ability toward Fe3+ 3 Cu2+ 4
, hypochlorous
,
acid,5 and volatile acidic gases.6 Rhodamine hydroxamic acid
derivatives that are tethered to an alkyne or triazole moiety have
been developed for the detection of Au3+ or Pt2+ 7,8
Yang et al.
.
reported that rhodamine hydroxamic acid with a 2-deoxyribose
moiety could selectively detect cysteine and homocysteine in
water.9 Additionally, the Lossen rearrangement of rhodamine
hydroxamic acid was applied to the detection of a nerve agent sim-
ulant, diethyl chlorophosphate.10 However, despite the potential
Hg2+ sensing ability of hydroxamic acid derivatives, no studies
regarding Hg2+-signaling have yet been conducted.
The replacement of an oxygen atom with a sulfur atom in a
specific ligand system is often a powerful tool for the fine control
of the metal affinity of the ligands.11 Previously, the thionation of
Hg2+ signaling is crucial from both an industrial and ecological
viewpoint because of its high impact on the environment.20
Many intricate sensors and reaction-based probes are being con-
tinuously developed for the signaling of this important species.21
Among the many signaling motifs, rhodamine derived reaction-
based probes have been particularly successful and attractive
because of their marked chromogenic and fluorogenic responses
⇑
Corresponding author. Tel.: +82 2 820 5199; fax: +82 2 825 4736.
E-mail address: skchang@cau.ac.kr (S.-K. Chang).
http://dx.doi.org/10.1016/j.tetlet.2015.06.085
0040-4039/Ó 2015 Elsevier Ltd. All rights reserved.

4920
Y. A. Jeong et al. / Tetrahedron Letters 56 (2015) 4919–4922
toward Hg2+ species.22 A number of derivatives based on rho-
damine hydrazide frameworks with an extra binding site have
been reported for Hg2+ signaling.23 However, rhodamine hydrox-
amic acid, which is another important rhodamine lactam platform,
has not been tested because of its relative inertness toward most
metal ions. We anticipated that the transformation of hydroxamic
acid functionality into its thio-analogue by thionation would
change the molecule into a probe with metal ion signaling
responses.
conditions (Figs. 1 and S1, Supplementary data). The UV–vis spec-
trum of 1 revealed an intense absorption band at 574 nm exclu-
sively in the presence of Hg2+ ions. This absorbance enhancement
was very large (the absorbance ratio in the presence and absence
of metal ions, A/A0 at 574 nm = 550) because of the characteristic
opening of the spirolactam moiety of rhodamine 1. The other metal
ions induced almost no response (A/A0 ranged from 0.1 to 3.2). In
particular, the interference of other thiophilic metal ions, including
Ag+ and Cu2, + was insignificant (the absorbance ratio in the presence
and absence of metal ions at 574 nm, A/A0, was 19 for Cu2+ and 5.5
for Ag+).
The fluorescence signaling behaviors of 1 and 2 were measured
in the same 50% aqueous DMSO solution. The parent hydroxamic
acid 2 did not respond to any surveyed metal ions (Fig. S2,
Supplementary data). In contrast, a prominent Hg2+-selective fluo-
rescence signaling was observed for the thio-analogue 1 (Figs. 2,
and S3, Supplementary data). The fluorescence enhancement at
600 nm was larger than 950 fold (U1+Hg(II) = 0.11).25 The other
metal ions only had minor responses and the fluorescence inten-
sity ratios in the presence and absence of metal ions I/I0 of 1 at
600 nm varied within a narrow range between 0.8 for Na+ and 12
for Ag+ ions.
Herein, we developed a new Hg2+-selective probe by controlling
the metal ion affinity of rhodamine hydroxamic acid by converting
it into its thio-functionalized analogue. Through the introduction
of a thio functional group into the hydroxamic acid, we obtained
a new rhodamine-based probe with improved responses and
marked sensitivity toward Hg2+ ions.
The thio derivative of rhodamine hydroxamic acid, 1, was pre-
pared by the thionation of hydroxamic acid, 2, which was obtained
using a literature procedure consisting of the reaction of rho-
damine B base with hydroxylamine (NaOH),10 using Lawesson’s
reagent (65%) (Scheme 1).24 In fact, the thio-analogue of rho-
damine hydroxamic acid was known, but its signaling behavior
toward any analytes has not been studied.14 Compound 1 has no
absorption bands above 480 nm and very weak emissions because
it is in its ring-closed thiolactam form (quantum yield
U1 = 0.0002).25
The possible interference of the Hg2+ signaling of 1 by the pres-
ence of coexisting metal ions was measured. The Hg2+-selective sig-
naling behavior of 1 was not strongly affected by the presence of
other commonly encountered metal ions (Fig. 3). The variation of
fluorescence intensity of the 1–Hg2+ system at 600 nm in the pres-
ence and absence of coexisting metal ions I(1+Metal ions+Hg(II))/I(1+Hg(II))
under competitive conditions ([1]:[Hg2+]:[Mn+] = 1:50:100) fluctu-
ated only within a narrow range between 0.86 for Ag+ and 0.97
for Li+ ions.
O
O
NH2-OH HCl
N
OH
O
NaOH / Reflux
EtOH-H2O (1:1, v/v)
N
O
N
N
O
N
2
The Hg2+-selective signaling resulted from the Hg2+-induced
transformation of the hydroxythiolactam moiety of 1 into a nitrile
(Scheme 2). Similar transformation of Hg2+-induced conversion of
rhodamine-thiolactam to rhodamine nitrile and hypochlorous
acid-promoted oxidative transformation of 7-hydroxycoumarin-
based oxime to its nitrile analogue has been reported.26,27
Signaling is believed to proceed via initial formation of ring-
opened thiohydroxamic acid that subsequently converted to fluo-
rescent cyanide derivative with the concomitant elimination of
HgS and water molecule. The suggested transformation was sup-
ported by NMR, IR, and mass spectrum measurements. The mass
spectrum obtained for the signaling product of 1 in the presence
of 10 equiv of Hg2+ ions revealed a diagnostic peak at m/z = 424.5
corresponding to nitrile 3 (m/z = 424.2 calculated for C28H30N3O)
(Fig. S4, Supplementary data). IR measurements revealed a charac-
teristic nitrile band at 2233 cmÀ1 for 3 (Fig. S5, Supplementary
S
N
OH
Lawesson's reagent
Toluene / Reflux
2
N
O
N
1
Scheme 1. Synthesis of rhodamine B thiohydroxamic acid 1.
The metal ion signaling behavior of 1 was preliminarily studied
using UV–vis spectroscopy in a mixed solution of aqueous acetate
buffer (pH 4.8) and DMSO (1:1, v/v). Hydroxamic acid 2 did not
induce any changes in absorption behavior toward any of the sur-
veyed representative alkali (Na+, K+), alkaline earth (Mg2+, Ca2+), or
transition metal ions (Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Ag+, Cd2+, Pb2+
,
Hg2+). However, the thio-analogue, 1, exhibited a prominent
Hg2+-selective signaling behavior under the same experimental
0.6
20
1 + Hg2+
0.5
1 + Hg2+
16
12
8
0.4
0.3
0.2
1 + Cu2+
4
1 only
0.1
0
1 only
1 + representative metal ions
1 + representative metal ions
0
480
530
580
630
550
600
650
700
750
Wavelength (nm)
Wavelength (nm)
Figure 1. Changes in the UV–vis spectrum of 1 in the presence of various metal
ions. [1] = 5.0 Â 10À6 M and [Mn+] = 5.0 Â 10À4 M. In acetate buffered (pH 4.8, final
concentration = 10 mM) 50% aqueous DMSO solution.
Figure 2. Changes in the fluorescence spectrum of 1 in the presence of various
metal ions. [1] = 5.0 Â 10À6 M and [Mn+] = 5.0 Â 10À4 M. In acetate buffered (pH 4.8,
final concentration = 10 mM) 50% aqueous DMSO solution. kex = 540 nm.

Y. A. Jeong et al. / Tetrahedron Letters 56 (2015) 4919–4922
4921
1.2
1
0.8
0.6
0.4
0.2
0
Figure 4. Partial 1H NMR spectra of 1 and signaling product of 1 with Hg2+ in CDCl3.
[1] = 10 mM and [Hg2+] = 50 mM. In acetate buffered (pH 4.8, 10 mM) 50% aqueous
acetonitrile solution. The signaling mixture was extracted using dichloromethane
and was purified by column chromatography.
+ 1 + Hg2+
Figure 3. Competitive fluorescence signaling of Hg2+ by 1 in the presence of
commonly coexisting metal ions. [1] = 5.0 Â 10À6 M, [Hg2+] = 2.5 Â 10À4 M, and
[Mn+] = 5.0 Â 10À4 M. In acetate buffered (pH 4.8, 10 mM) 50% aqueous DMSO
solution. kex = 540 nm. The fluorescence intensity was measured at 600 nm.
20
20
15
10
5
[Hg2+
]
16
12
8
Hg2+
Hg+ X-
S
S
Hg2+
0
OH
OH
N
N
0
10
20 30
40 50
Et2N
O
NEt2
Et2N
O
NEt2
Equiv. of Hg2+
Colorless and
H+
Fluorescence OFF
4
Hg+ X-
0
S
550
600
650
Wavelength (nm)
700
750
C
N
- HgS
- H2O
OH2
N
Et2N
O
NEt2
Et2N
O
NEt2
X-
Figure 5. Fluorescence titration of
1
with Hg2+
.
[1] = 5.0 Â 10À6 M. In acetate
Pink colored and
buffered (pH 4.8, final concentration = 10 mM) 50% aqueous DMSO solution. The
inset shows the fluorescence intensity enhancement at 600 nm as a function of
[Hg2+]. kex = 540 nm.
Fluorescence ON
Scheme 2. Plausible signaling mechanism of Hg2+ ions by probe 1.
obtained
using
fluorescence
measurements
(Fig.
S9,
data). The 1H and 13C NMR spectra of the Hg2+ signaling product of
1 showed resonances that were expected for the postulated struc-
ture of nitrile 3 (Figs. 4 and S6, Supplementary data). The signaling
was irreversible as demonstrated by the effect of the addition of
EDTA to the 1–Hg2+ signaling system (Fig. S7, Supplementary data).
Upon treatment of 1 with 100 equiv of Hg2+ ions, the aforemen-
tioned chromogenic and fluorogenic signals were observed.
However, upon treatment of the resulting solution with EDTA
(2 equiv with respect to the added Hg2+), the observed signals were
not affected. This observation implied that the signaling of the 1–
Hg2+ system is an irreversible process.
Supplementary data). Compound 1 itself was stable and no notice-
able changes in fluorescence were observed even 5 h after sample
preparation.
Finally, the application of Hg2+-selective signaling behavior of 1
was tested using test strip experiments. Test strips were prepared
by impregnating filter paper (Whatman No. 2) with an acetonitrile
solution of 1. Upon treatment of the test strips with increasing
amount of Hg2+
, distinct pink-colored spots were observed
(Fig. 6). The color of the developed spots was analyzed using
Adobe Photoshop (Figs. S10 and S11, Supplementary data). As
shown in Figures S10 and S11, the developed color was well corre-
lated to the [Hg2+] of the analytes. This observation suggested that
the test strips could be used for the rapid and semi-quantitative
visualization of Hg2+ in aqueous environments.
The quantitative signaling behavior of 1 for Hg2+ ions was
assessed using a fluorescence titration (Fig. 5). The fluorescence
spectrum of 1 steadily increased as the concentration of Hg2+
increased up to 1.0 Â 10À4 M. Using this concentration dependent
absorption change, the detection limit for the determination of
Hg2+ by 1 was estimated to be 1.0 Â 10À6 M.28
In summary, a new Hg2+-selective optical probe was developed
through the thionation of rhodamine hydroxamic acid. The thiohy-
droxamic acid derivative had a pronounced dual signaling behavior
The Hg2+ signaling of 1 was found to be pH dependent (Fig. S8,
Supplementary data). As the pH of the solution increased from 4.8
to 8.0, the Hg2+ signaling slowly decreased, and after pH 8.0 no
detectable signal was observed. However, the spectral change of
1 alone was not observed over the entire pH region from 4.8 to
9.0. This profile may be due to the involvement of protons in the
postulated Hg2+ signaling mechanism (Scheme 2). At high pH
region, the concentration of proton which is crucial for the activa-
tion of thiohydroxamic acid function is low that is unfavorable for
the transformation to nitrile. From this observation, we confirmed
that the Hg2+ signaling of 1 was relatively optimized using an acet-
ate buffer at pH 4.8. On the other hand, the Hg2+-selective signaling
of 1 was complete within 5 min as monitored by a time trace plot
Figure 6. Pictures of the developed spots of 1 on test strips under (a) daylight and
(b) UV light illumination in the presence of varying [Hg2+]. [Hg2+] = 0–4.0 Â 10À3 M.

4922
Y. A. Jeong et al. / Tetrahedron Letters 56 (2015) 4919–4922
toward Hg2+ in the presence of other metal ions, which included
thiophilic Cu2+ and Ag+ ions. Tuning the structure of rhodamine
hydroxamic acid by thionation of the hydroxamic acid lactam moi-
ety resulted in a remarkably selective and effective signaling
behavior. The reported results provide useful information for the
development of Hg2+ probes by fine-tuning probe structures using
simple thionation.
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Acknowledgment
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This research was supported by the Korea Research Foundation
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Supplementary data associated with this article can be found, in
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