A rhodamine-based Hg2+ sensor with high selectivity and sensitivity in aqueous solution: A NS2-containing receptor

Article abstract of DOI:10.1021/jo802297x

A rhodamine-based sensor 1 was designed and synthesized by incorporation the rhodamine fluorophore and ionophore NS₂ with high affinity to Hg²⁺. Sensor 1 exhibits a high selectivity and an excellent sensitivity and is a dualresponsiv

Full text of DOI:10.1021/jo802297x

A Rhodamine-Based Hg2+ Sensor with High
Selectivity and Sensitivity in Aqueous Solution:
A NS2-Containing Receptor
SCHEME 1. Representative Mechanism of the
Chemosensor Based on the RhB
Junhai Huang, Yufang Xu, and Xuhong Qian*
State Key Laboratory of Bioreactor Engineering, Shanghai
Key Laboratory of Chemical Biology, School of Pharmacy,
East China UniVersity of Science and Technology,
Shanghai 200237, China
xhqian@ecust.edu.cn
spirocyclic moiety mediated by guests as shown in Scheme 1.2-9
When guests are bound to the sensors, the spirocyclic form of
RhBs, which is colorless and nonfluorescent, is converted to
the opened-cyclic form which is pink and strongly fluorescent.2-9
However, this conversion is strongly dependent on the organic
solvent content or pH value in detecting solution system.2-9
For the known reversible sensors based on the rhodamine
moiety, most of them work well in a pure organic solvent media
(such as MeCN2,3c,5d,e,6c,e or MeCN/methanol6a) or an aqueous
solution containing at least 50% organic cosolvent (such as
DMF,5f ethanol,5b,6b,7a,b methanol,4c or MeCN3b,d). Also, some
of them work well in strong acidic solution at pH 3-45g,h,10 or
strong basic solution at pH 12.8b Moreover, this conversion will
reverse when some competitive solvents, such as water, are
added into the sensing system. These limitations, including
organic cosolvent dependence and pH dependence, to some
extent, lower the sensitivity and restrict the application of
rhodamine-based sensors in biological systems and environ-
mental determinations.11 In fact, only a few of them, particularly
in irreversible rhodamine-based ion sensors,4a,b,d work well in
ReceiVed October 13, 2008
A rhodamine-based sensor 1 was designed and synthesized
by incorporation the rhodamine fluorophore and ionophore
NS2 with high affinity to Hg2+. Sensor 1 exhibits a high
selectivity and an excellent sensitivity and is a dual-
responsive colorimetric and fluorescent Hg2+-specific sensor
in aqueous buffer solution. In addition, the 1:1 binding mode
was proposed based on the 1H NMR and ES(+)MS studies.
(4) (a) For Hg2+ (irreversible sensor): Yang, Y. K.; Yook, K. J.; Tae, J. J. Am.
Chem. Soc. 2005, 127, 16760–16761. (b) Shi, W.; Ma, H. Chem. Comm. 2008,
16, 1856–1858. (c) Wu, J.; Hwang, I.; Kim, K.; Kim, J. Org. Lett. 2007, 9,
907–910. (d) Zhang, X.; Xiao, Y.; Qian, X. Angew. Chem., Int. Ed. 2008, 47,
8025–8029.
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Ch.; He, Ch.; Wu, Sh.; Wang, D. Inorg. Chem. 2008, 47, 7190–7201. (b) Yang,
H.; Zhou, Z.; Huang, K.; Yu, M.; Li, F.; Yi, T.; Huang, C. Org. Lett. 2007, 9,
4729–4723. (c) Suresh, M.; Shrivastav, A.; Mishra, S.; Suresh, E.; Das, A. Org.
Lett. 2008, 10, 3013–3016. (d) Lee, M.; Wu, J.; Lee, J.; Jung, J.; Kim, J. Org.
Lett. 2007, 9, 2501–2504. (e) Soh, J.; Swamy, K.; Kim, S.; Kim, S.; Lee, S.;
Yoon, J. Tetrahedron Lett. 2007, 48, 5966–5969. (f) Wu, D.; Huang, W.; Duan,
C.; Lin, Z.; Meng, Q. Inorg. Chem. 2007, 46, 1538–1540. (g) Zheng, H.; Qian,
Z.; Xu, L.; Yuan, F.; Lan, L.; Xu, J. Org. Lett. 2006, 8, 859. (h) Zhan, X.; Qian,
Z.; Zheng, H.; Su, B.; Lan, Z.; Xu, J. Chem. Commun. 2008, 16, 1859–1861.
(6) (a) For Fe3+: Bae, S.; Tae, J. Tetrahedron Lett. 2007, 48, 5389–5392.
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Liu, W. Org. Lett. 2007, 9, 4567–4570.
Rhodamine B and its derivatives (RhBs) are well-known for
their desirable properties, including good photostability, high
extinction coefficient (>75000 cm-1 M-1), and high fluores-
cence quantum yield, particularly in its nucleotide and nucleic
acid conjugates.1 Recently, rhodamine-based sensors for cations
and other analytes have received ever-increasing interest in areas
such as for sensors for Pb2+, Cu2+, Hg2+, Fe3+, Cr3+, NO, and
OCl-.2-8 The mechanism is based on the switch off/on of the
(7) (a) For Cr3+: Zhou, Z.; Yu, M.; Yang, H.; Huang, K.; Li, F.; Yi, T.;
Huang, Ch. Chem. Comm. 2008, 16, 3387–3389. (b) Huang, K.; Yang, H.; Zhou,
Z.; Yu, M.; Li, F.; Gao, X.; Yi, T.; Huang, Ch. Org. Lett. 2008, 10, 2557–2560.
(8) (a) For NO and OCl: Zheng, H.; Shang, G.; Yang, S.; Gao, X.; Xu, J.
Org. Lett. 2008, 10, 2357–2360. (b) Chen, X.; Wang, X.; Wang, S.; Shi, W.;
Wang, K.; Ma, H. Chem.sEur. J. 2008, 14, 4719–4724.
(9) Kim, H.; Lee, M.; Kim, H.; Kim, J.; Yoon, J. Chem. Soc. ReV. 2008, 37,
1465–1472.
(10) Proton could induce the opened-cycle of spirolactam below pH5.0;
therefore, the acid media was not suitable to determine targeted-analytes.
(11) In general, the sensor for biological application works in near neutral
conditions.
(1) Haugland, R. P. The Handbook: a guide to fluorescent probes and labeling
technologies, the tenth edition, molecular probes; Invitrogen Corp.: Karlsbad,
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(2) (a) For Pb2+: Kwon, J. Y.; Jang, Y. J.; Lee, Y. J.; Kim, K. M.; Seo,
M. S.; Nam, W.; Yoon, J. J. Am. Chem. Soc. 2005, 127, 10107–10111.
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5042. (d) Lee, M. H.; Kim, H. J.; Yoon, S.; Park, N.; Kim, J. S. Org. Lett. 2008,
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10.1021/jo802297x CCC: $40.75
Published on Web 02/11/2009
2009 American Chemical Society
J. Org. Chem. 2009, 74, 2167–2170 2167

SCHEME 2. Synthesis of 1
aqueous buffer solution containing less than 20% organic
cosolvent8a or in neutral pure water.5a,f To solve this problem,
the simplest method is to introduce an appropriate receptor R
(Scheme 1) which has several features, including (1) reversible
binding to guest molecules, (2) high affinity to guest molecules,
and (3) conformational preorganization to facilitate capture of
guest molecules.12 Obviously, the receptor introduced should
improve the ability to competitively bind the guest molecules
in aqueous buffer solution and reduce the amount of organic
cosolvent in the detecting solution.
Herein, we designed a new rhodamine B-based chemosensor
13
1 for Hg2+
.
In 1, the receptor contained the NS2 fragment,
FIGURE 1. (a) Absorption spectra of 1 (5 µM) in buffer solution upon
addition of different amounts of Hg2+ ions. Inset: the Job’s plot; the
total concentration of ([Hg2+] + [1]) was 10 µM. The photograph shows
the color change of 1 (5 µM) in solution. (b) Fluorescence spectra of
1 (5 µM) under the same conditions upon addition of different amounts
of Hg2+ ion. Excitation was performed at 530 nm. Inset: fluorescence
enhancement at 597 nm as a function of Hg2+ concentration. The
photograph shows the fluorescent color of 1 (5 µM) upon addition 2.0
equiv Hg2+ in solution with 365 nm excitation.
which is a well-known specific and reversible binding receptor
of Hg2+ due to the thiophilic nature of mercury and has been
used in a fluorescein-based PET sensor.13a Therefore, we
speculated that the introduction of the NS2 receptor to a
rhodamine-based probe would (1) increase the affinity to Hg2+
in competitive aqueous media, (2) quickly induce the fluorescent
and color responses, that is, realize the real-time detection, (3)
improve the selectivity, and (4) recognize Hg2+ reversibly.
Compound 1 was synthesized and characterized by NMR in
addition to mass data. The preliminary experiments showed that
1 displayed a high selectivity and sensitivity for Hg2+ in buffer
solution. The bind mode was proposed by the 1H NMR titration
study and ES(+)-MS analysis.
The acid-base titration experiments revealed that 1 did not emit
any obvious and characteristic (excitation at 530 nm) fluores-
cence in the pH range from 6.0 to 12.0, suggesting that it was
insensitive to pH near 7.0 and could work in approximate
physiological conditions with
a very low background
NS2 was synthesized according to the published procedure.13a
Compound 1 was synthesized by treating rhodamine B with
POCl3, which was followed by NS2. After column chromatog-
raphy using DCM/MeOH (100/3, v/v) as eluent, sensor 1 was
obtained in a 26% yield (Scheme 2).
fluorescence.13a Therefore, further UV/vis and fluorescent studies
were carried out in MeCN/HEPES mixed buffer solution
(MeCN/water ) 15/85, v/v, pH 6.98, 20 mM HEPES, 50 mM
KCl).
Like most of the spirocycle RhB derivatives, the free 1
remained colorless and did not exhibit apparent absorption above
500 nm in the above buffer system (as shown in Figure 1). This
indicated that the spirolactam form (Scheme 1) was the
predominant species.2-8 Upon addition of Hg2+ ion, a new
strong absorption band centered at 573 nm was formed and led
to the color change from colorless to purple. This indicated that
the opened-ring form (Scheme 1) of 1 became the main species
in the examined solution.2-8 The emission spectra were also
recorded under the same conditions. Free 1 displayed a very
weak fluorescence. When Hg2+ ion was added to the buffer
solution of 1, a significant increase (almost 400-fold enhance-
ment of IF/I0, herein I0, indicated the fluorescence intensity of
free 1; IF indicated the fluorescence intensity upon adding 2.0
equiv Hg2+) of fluorescence at 597 nm was observed, that is,
Hg2+ ion induced the formation of open-cycle RhBs with strong
fluorescence (Scheme 1).2-8 The 1:1 stoichiometry between 1
and Hg2+ was confirmed by the Job’s plot shown in the inset
of Figure 1a. The binding constant Ka was (1.18 ( 0.13) × 106
The pH response of 1 in MeCN/water solution (2/8, v/v) was
first evaluated as shown in Figure S3, Supporting Information.
(12) Pfeffer, F.; Seter, M.; Lewcenko, N.; Barnett, N. Tetrahedron Lett. 2006,
47, 5241–5245.
(13) (a) Some examples of Hg2+ ion fluorescent probe and reviews: Nolan,
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Lippard, S. J. J. Am. Chem. Soc. 2007, 129, 5910–5918. (d) Descalzo, A. B.;
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3418–3419. (e) Wang, J.; Qian, X.; Cui, J. J. Org. Chem. 2006, 71, 4308–4311.
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McCoy, C. P.; Rademacher, J. T.; Rice, T. E. Chem. ReV. 1997, 97, 1515–1566.
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2168 J. Org. Chem. Vol. 74, No. 5, 2009

FIGURE 2. Fluorescent and absorption spectra of 1 (5 µM) in the
presence of different metal ions: (a) fluorescent spectra, Hg2+ (2.0 equiv)
and other ions (10.0 equiv), excitation was performed at 530 nm; (b)
UV/vis spectra, Hg2+ (2.0 equiv) and other ions (10.0 equiv).
M-1, inferred from the Hg2+ titration curves (Figure S4,
Supporting Information).14 In addition, the EDTA-adding
experiments were conducted to examine the reversibility of this
reaction as shown in Figure S5 (Supporting Information). When
EDTA was added to the solution of equimolar 1/Hg2+, the color
changed from pink to colorless and the fluorescence was turned
off. These results indicated that 1 was a reversible chemosensor
for Hg2+; a similar result was reported in a sensor with the same
receptor.13a
FIGURE 3. Time evolution of sensor 1 (5 µM) in MeCN/water (15/
85, v/v) buffer (pH6.98, 20 mM HEPES, 50 mM KCl) in the presence
of 2.0 equiv of Hg2+ ion. Inset: changes of fluorescence intensity at
597 nm as a function of time (0.0 to 10.0 min). Excitation was
performed at 530 nm.
To validate the selectivity of 1 in practice, some other cations
were added to a solution of 1 under the same conditions. The
various alkali, alkaline earth metal ions, and transition metal
ions (Pb2+, Cu2+, Cd2+, Fe3+, Fe2+, Zn2+, Cr3+, Co2+, and Ni2+)
did not induce any apparent fluorescent enhancement and color
change even upon addition of 10 equiv of the respective metal
ions (as shown in Figure 2 and Figure S6, Supporting Informa-
tion). The ratio of IF/I0 at 597 nm (here, I0 indicates the
fluorescence intensity of free 1 and IF indicates the fluorescence
intensity upon addition of different metal ions) of 1 in the
presence of various other metal ions is shown in Figure S6a
(Supporting Information). For Hg2+, the IF/I0 value was almost
400-fold, while the values for the other metal ions were less
than 10-fold. Therefore, 1 was a highly selective chemosensor
for Hg2+. The competitive experiments were conducted in the
presence of 2.0 equiv of Hg2+ mixed with 10.0 equiv of various
cations, respectively. No significant variation in fluorescence
intensity was found by comparison with that without other metal
ions besides Hg2+ (Figure S6b, Supporting Information). These
results indicated that 1 was a Hg2+-specific fluorescent sensor.
As a sensor, real-time determination was necessary. We next
investigated the time evolution of the responses of 1 (5 µM) in
the presence of 2.0 equiv of Hg2+ in same buffer solution. As
shown in Figure 3, the recognition interaction was completed
immediately after addition of the Hg2+ without any detectable
time-delay. Compared to its analogues (which need a equilib-
rium time before detection3a,5a,d), therefore, sensor 1 was a
sensitive sensor and could be used in real-time determination
of Hg2+ in environmental analysis.
FIGURE 4. (a) Hg2+ 1H NMR titration of 1 (10.0 mM) in CD3CN
and CD3OD (1:1): (a) 1 only; (b) 1 + 0.25 equiv of Hg2+; (c) 1 + 0.50
equiv of Hg2+; (d) 1 + 0.75 equiv.of Hg2+; (e) 1 + 1.00 equiv of
Hg2+
.
- +
ClO4 ] (the calculated value is 1023.3). To further elucidate
the binding mode, the 1H NMR-titration experiments were
conducted. As shown in Figure 4 and Figure S8 (Supporting
Information), a set of new peaks appeared with the increase of
Hg2+ ion in the range of 0.0-1.0 equiv, while there was no
further change after addition of more than 1.0 equiv of Hg2+
,
which further confirmed the 1:1 stoichiometry. That H5 and H6
beside the “S” group displayed an apparent downfield shift (from
peak centered at 3.33 ppm to 3.63 ppm, the ∆δ ) 0.30 ppm,
upon addition of 1.0 equiv of Hg2+ ion) originated from the
coordination “S” to “Hg2+”. H4 displayed a similar downfield
shift (from peak centered 2.40 ppm to 2.68 ppm, the ∆δ )
0.28 ppm), and it suggested that Hg2+ bound to the “N” and
induced the decrease of the electronic density in H4. H7 (the
signal of H7 was overlapped with H2) also showed an apparent
downshift. In addition, the downshift of H1 and H2 clearly
suggested that the Hg2+ induced the formation of delocalized
xanthene moiety of the rhodamine, which increased the deshield-
ing effect and caused the H1 and H2 downshift of 0.28 and 0.17
ppm, respectively. The downshift of peaks near δ6.0 ppm, which
were assigned to the proton signal of xanthene, also proved the
delocalization of xanthene; that is, Hg2+ ion induced the opening
of spirocycle. Taken together, the above results indicate a
plausible interaction mode of 1/Hg2+ as proposed in Figure 5,
in which Hg2+ was coordinated with two “S”, “N”, and carbonyl
“O”.
As mentioned above, the 1:1 coordination mode was con-
firmed by the Job’s plot, which was also supported by ES(+)-
MS.15 Without Hg2+ ion, the peak m/z 723.2 corresponds to [1
+ H]+. When 1.0 equiv of Hg2+ is introduced to a 1 solution,
a peak appears at m/z 1023.3 (Figure S7, Supporting Informa-
tion) and is assigned to single-charged complex [1 + Hg2+
+
(14) Valeur, B. Molecular Fluorescence. Principles and Applications; Wiley-
VCH: Weinheim, 2002.
(15) (a) The ESI experiment was carried out in MeCN-water (1/9, v/v)
solution considering the interference of HEPES salts: 1 (2 µM); Hg2+ (2 µM).
(b) ESI(+) was extensively used to determine the complexe ration between ligand
and metal ion: Pratesi, A.; Zanello, P.; Giorgi, G.; Messori, L.; Laschi, F.; Casini,
A.; Corsini, M.; Gabbiani, C.; Orfei, M.; Rosani, C.; Ginanneschi, M. Inorg.
Chem. 2007, 46, 10038–10040.
J. Org. Chem. Vol. 74, No. 5, 2009 2169

oxychloride (0.4 mL) was added dropwise over 2 min. The solution
was refluxed for 2 h. The reaction mixture was cooled and
evaporated in vacuo to give rhodamine B acid chloride, which was
not purified and used in the next step directly. The crude acid
chloride was dissolved in acetonitrile (80 mL) and added dropwise
over 1 h to a solution of 2 (0.30 g, 1.0 mmol) and TEA (0.5 mL)
in acetonitrile (20 mL) at room temperature. The reaction mixture
was then refluxed for 1 h. After the solvent was evaporated under
reduced pressure, the crude product was purified by column
chromatography (DCM/MeOH, 100:3, v/v) to give 188 mg of 1
1
(yield 26%): mp 77-79 °C; H NMR (CD3CN) δ 1.08-1.68 (m,
FIGURE 5. Proposed binding mode between 1/Hg2+
.
18H), 2.32-2.46 (m, 12H), 2.67 (d, 1H, J ) 16.0 Hz), 3.25 (d,
1H, J ) 16.0 Hz), 3.28-3.40 (m, 8H), 6.00 (d, 1H, J ) 4.0 Hz),
6.15 (d, 1H, J ) 2.4 Hz), 6.25 (d, 1H, J ) 2.4 Hz), 6.36 (dd, 1H,
J1 ) 8.8 Hz, J2 ) 2.8 Hz), 6.49 (dd, 1H, J1 ) 8.8 Hz, J2 ) 2.4
Hz), 6.57 (t, 2H, J ) 8.4 Hz), 6.87 (t, 1H, J ) 7.6 Hz), 7.19-7.25
(m, 2H), 7.63-7.07 (m, 3H), 7.92 (d, 1H, J ) 6.8 Hz); 13C NMR
(CD3CN) δ 11.7, 14.3, 11.9, 25.4, 29.1, 44.0, 44.1, 53.5, 53.9, 67.8,
96.8, 97.5, 106.4, 107.3, 107.6, 108.5, 116.8, 117.3, 122.8, 124.3,
126.1, 127.9, 128.3, 128.8, 128.9, 130.2, 132.5, 132.8, 134.4, 140.5,
149.2, 151.5, 153.9, 154.5, 165.6; HRMS (ESI+) found 723.3755
(M + H)+, calcd for C43H55N4O2S2 723.3766.
In conclusion, we report a rhodamine derivative 1 used as a
selective and sensitive chemosensor, which could specifically
recognize Hg2+ ion in aqueous buffer solution by the “naked
eye”, UV/vis, and fluorescent responses. Compared with the
reported rhodamine-based chemosenor, 1 reduced the amount
of organic cosolvent in detecting media and improved sensitivity
and selectivity through a rational incorporation of NS2 receptor
(NS2 has high affinity toward Hg2+ due to the thiophilic nature
of mercury) to the rhodamine structure. These results also
indicated that the introduction of receptor with high affinity to
targeted analytes in the rhodamine-based sensor would improve
the selectivity and sensitivity. In addition, the spectral res-
ponse toward Hg2+ was established to be reversible by the
EDTA-titration experiments. Furthermore, the 1:1 coordination
mode was proposed on the basis of the 1H NMR titration
experiments and ES(+)-MS analysis.
Acknowledgment. This work is supported by the National
Natural Science Foundation of China (20536010, 20746003),
Program of Shanghai Subject Chief Scientist and Shanghai
Leading Academic Discipline Project (B507), and the National
High Technology Research and Development Program of China
(863 Program 2006AA10A201).
Experimental Section
Supporting Information Available: Synthesis and charac-
1
terization of 1; HRMS, H NMR, and 13C NMR spectra and
Compound NS2. Synthesis according to reported method:13a
1H NMR (CDCl3) δ 1.20 (t, 6H, J ) 7.6 Hz), 2.45 (q, 4H, J ) 7.6
Hz), 2.62-2.66 (m, 4H), 2.69-2.73 (m, 4H), 3.65 (s, 2H), 4.75
(br, 2H), 6.62-6.67 (m, 2H), 6.98 (d, 1H, J ) 7.6 Hz), 7.08 (td,
1H, J1 ) 7.6 Hz, J2 ) 1.2 Hz); 13C NMR (CDCl3) δ 14.8, 25.9,
29.1, 53.3, 58.4, 115.6, 117.6, 122.4, 128.6, 130.4, 147.1.
the spectroscopic data; pH-titration of free 1; the curve fitting;
ESI(+)-MS experiment and spectrum in the presence of the
equimolar Hg2+/1 (2 µM); EDTA-titration of absorption and
emission; selective and competitive experiments. This material
is available free of charge via the Internet at http://pubs.acs.org.
Compound 1. A solution of rhodamine B base (0.45 g, 1.0
mmol) in 1,2-dichloromethane (15 mL) was stirred, and phosphorus
JO802297X
2170 J. Org. Chem. Vol. 74, No. 5, 2009