Detecting Hg2+ ions with an ICT fluorescent sensor molecule: Remarkable emission spectra shift and unique selectivity

Article abstract of DOI:10.1021/jo052642g

A fluorescent ratiometric Hg²⁺ ion sensor RMS, based on a coumarin platform coupled with a tetraamide receptor, is presented. This sensor, employing the ICT mechanism, could be used to specifically detect Hg ²⁺ ions in a neutral buff

Full text of DOI:10.1021/jo052642g

Detecting Hg2+ Ions with an ICT Fluorescent
Sensor Molecule: Remarkable Emission Spectra
Shift and Unique Selectivity
wavelength shift. We chose coumarin as the fluorophore of RMS
in consideration of its desirable photophysical properties such
as large Stokes shift and visible excitation and emission
wavelength.5,6 Another important qualification is the tunability
of photophysical properties of the coumarin family compounds.
In RMS, the fluorophore is a strong “push-pull” π-electron
system, with the two aniline nitrogen atoms as the electron
donor, and two electron-deficient groups, namely, one carbonyl
and one benzothiazolyl group, as the electron acceptor. The
fluorophore will undergo an intramolecular charge transfer
(ICT)7 from the donor to the acceptor upon excitation by light.
This excited state shows a long emission wavelength because
of the large extent of π-electron conjugation. However, when
the tetraamide receptor8 has caught a Hg2+ ion, the electron-
donating ability of the 6-, 7-nitrogen will be decreased, and thus
a blue shift in both absorption and emission spectra should be
observed, resulting from a reduction in the π-electron conjuga-
tion. RMS and the tetraester control sensor (CS) were prepared
by a series of steps starting from 4-amino-3-nitrophenol. The
tetraamide Hg2+ ion receptor (MR) was prepared starting from
o-phenylenediamine (Scheme 1).
Jiaobing Wang,† Xuhong Qian,*,†,‡ and Jingnan Cui†
State Key Laboratory of Fine Chemicals,
Dalian UniVersity of Technology, Dalian 116012, China, and
Shanghai Key Laboratory of Chemical Biology, School of
Pharmacy, East China UniVersity of Science and Technology,
Shanghai 200237, China
xhqian@ecust.edu.cn
ReceiVed December 23, 2005
UV-visible absorption spectra and emission spectra of RMS
did not exhibit any detectable change under pH ranging from
3.0 to 8.0 (ꢀ ) 24 000 M-1 cm-1, φ ) 0.051).9 When pH was
further decreased, a band centered at 490 nm formed and
developed. The solution color changed from yellow to orange.
A pKa value of 1.2 was derived from the pH titration curve.
Red shift of the absorption spectra and the presence of an
isosbestic point at 452 nm together indicated a new species
corresponding to the protonation of the benzothiazole nitrogen
on the coumarin fluorophore (Figure 1).10 This interpretation
was also supported by the gradual fluorescence quenching at
lower pH (see Supporting Information).
The Hg2+ ion titration experiment revealed several intriguing
features of RMS. The unbound RMS exhibited a maximum
absorption at 430 nm, which gradually shifted to short wave-
length with the sequential addition of Hg2+ ions (Figure 2),
illustrating that Hg2+ ion was caught by the tetraamide chelating
site of RMS and resulted in a reduction in the aniline nitrogens’
A fluorescent ratiometric Hg2+ ion sensor RMS, based on a
coumarin platform coupled with a tetraamide receptor, is
presented. This sensor, employing the ICT mechanism, could
be used to specifically detect Hg2+ ions in a neutral buffered
water solution with an ∼100-nm blue shift in emission
spectra.
It is appealing to selectively detect Hg2+ ions in water solution
with a highly sensitive fluorescent sensor molecule.1 Since the
analyte, as one of the most prevalent components in the mercury
family compounds (elemental, inorganic, and organic mercury),
plays an important role in mercury biogeochemical cycling and
mercury toxicology,2 closer monitoring of the Hg2+ ions will
help us to attain a thorough evaluation of the character and
process of mercury distribution and transformation.3
(4) Ratiometric fluorescent sensors have many inherent properties suitable
for detecting analytes. See: (a) Grynkiewicz G.; Poenie, M.; Tsien, R. Y.
J. Biol. Chem. 1985, 260, 3440. (b) Mello, J. V.; Finney, N. S. Angew.
Chem., Int. Ed. 2001, 40, 1536. (c) Choi, K.; Hamilton, A. D. Angew. Chem.,
Int. Ed. 2001, 40, 3912. (d) Takakusa, H.; Kikuchi, K.; Urano, Y.; Sakamoto,
S.; Yamaguchi, K.; Nagano, T. J. Am. Chem. Soc. 2002, 124, 1653.
(5) Trenor, S. R.; Shultz, A. R.; Love, B. J.; Long, T. E. Chem. ReV.
2004, 104, 3059.
(6) Lim, N. C.; Schuster, J. V.; Porto, M. C.; Tanudra, M. A.; Yao, L.;
Freake, H. C.; Bru¨ckner, C. Inorg. Chem. 2005, 44, 2018.
(7) (a) Valeur, B. Molecular Fluorescence: Principles and Applications;
Wiley-VCH: Weinheim, 2001; pp 337-350. (b) de Silva, A. P.; Gunaratne,
H. Q. N.; Gunnlaugsson, T.; Huxley, A. J. M.; McCoy, C. P.; Rademacher,
J. T.; Rice, T. E. Chem. ReV. 1997, 97, 1515.
(8) Wang, J.; Qian, X. Chem. Commun. 2006, 109. Recently, we have
disclosed that o-phenylenediamine-based triamide receptor could be used
to selectively bind Hg2+ ions in water solution.
Herein, we report a novel fluorescent ratiometric4 Hg2+ ions
sensor (RMS) that could be used to selectively detect Hg2+ ions
in a neutral buffered water solution with a significant emission
† Dalian University of Technology.
‡ East China University of Science and Technology.
(1) For recent examples, see: (a) Ros-Lis, J. V.; Marcos, M. D.; Mart´ınez-
ma´n˜ez, R.; Rurack, K.; Soto, J. Angew. Chem., Int. Ed. 2005, 44, 4405. (b)
Coronado, E.; Gala´n-Mascaros, J. R.; Mart´ı-Gastaldo, C.; Palomares, E.;
Durrant, J. R.; Vilar, R.; Gratzel, M.; Nazeeruddin, Md. K. J. Am. Chem.
Soc. 2005, 127, 12351. (c) Guo, X.; Qian, X.; Jia, L. J. Am. Chem. Soc.
2004, 126, 2272. (d) Ono, A.; Togashi, H. Angew. Chem., Int. Ed. 2004,
43, 4300. (e) Nolan, E. M.; Lippard, S. J. J. Am. Chem. Soc. 2003, 125,
14270. (f) Descalzo, A. B.; Mart´ınez-ma´n˜ez, R.; Radeglia, R.; Rurack, K.;
Soto, J. J. Am. Chem. Soc. 2003, 125, 3418.
(2) Tchounwou, P. B.; Ayensu, W. K.; Ninashvili, N.; Sutton, D. EnViron.
Toxicol. 2003, 18, 149.
(3) Zhang, Z.; Guo, X.; Qian, X.; Lv, Z.; Liu, F. Kidney Int. 2004, 66,
2279.
(9) Determined relative to quinine sulfate. Meech, S. R.; Phillips, D. C.
Photochemistry 1983, 23, 193.
(10) 1H NMR studies also indicate that protonation occurs at the
benzothiazole nitrogen since proton Ha (Figure 1, inset) near the ring
nitrogen undergoes a significant downfield shift (0.4 ppm) upon addition
of hydrochloric acid (see Supporting Information). Corrent, S.; Hahn, P.;
Pohlers, G.; Connolly, T. J.; Scaiano, J. C.; Forne´s, V.; Garc´ıa, H. J. Phys.
Chem. B 1998, 102, 5852.
10.1021/jo052642g CCC: $33.50 © 2006 American Chemical Society
Published on Web 04/29/2006
4308
J. Org. Chem. 2006, 71, 4308-4311

SCHEME 1. Synthesis of Compounds RMS, MR, and CSa
FIGURE 1. pH effect on the UV-visible absorption of RMS. Inset:
structure of the protonated fluorophore, A490/A430 as a function of
solution pH, and the visualized solution color change.
a Reagent: (a) benzyl bromide, NaOH, tetrabutylammonium bromide,
H2O-CH2Cl2; (b) SnCl2‚2H2O, acetonitrile ethanol; (c) ethyl bromoacetate,
N,N-diisopropylethylamine, acetonitrile; (d) DMF, POCl3; (e) Pd/C (10%),
cyclohexene, tetrahydrofuran; (f) 2-aminoethanol, acetonitrile; (g) CF3COOH,
methanol; (h) piperidine, methanol; (i) piperidine, ethanol.
ability to participate in π-electron conjugation. The solution
color changed from yellow to almost colorless in the presence
of 40 equiv of Hg2+ ions. A clear isosbestic point at 390 nm
indicated the coexistence of the free RMS and the Hg2+-RMS
complex.
A Job’s plot (see Supporting Information) indicated that RMS
formed a 1:1 complex with Hg2+ ion in water solution.11 The
association constant log Ks, derived from the titration curve,
was 4.41 ( 0.02.
FIGURE 2. Absorption (top, inset: A430/A370 as a function of Hg2+
ion concentration and the visualized solution color change) and emission
(bottom, inset: I567/I475 as a function of Hg2+ ion concentration, λex
)
390 nm) spectra of RMS in the presence of different concentrations of
Hg2+ ions. Condition: 5 µM RMS in 0.05 M phosphate-buffered water
solution (pH ) 7.5).
When excitation was at 390 nm, emission spectra revealed a
significant blue shift with the gradual increasing of Hg2+ ion
J. Org. Chem, Vol. 71, No. 11, 2006 4309

FIGURE 4. Fluorescence response of RMS in the presence of 20 equiv
of different metal ions. Conditions: 10 µM RMS in 0.05 M HEPES13-
buffered water solution (pH ) 7.5). 0, control. 1, Cd2+. 2, Hg2+. 3,
Fe3+. 4, Zn2+. 5, Ag+. 6, Co2+. 7, Cu2+. 8, Ni2+. 9, Pb2+. The salts
employed are Cd(NO3)2‚4H2O, HgCl2, Fe(NO3)3, Zn(NO3)2, AgClO4‚
H2O, CoCl2‚6H2O, Cu(ClO4)2‚6H2O, NiCl2‚6H2O, and Pb(ClO4)2‚3H2O,
respectively.
FIGURE 3. Modulating the fluorescence of RMS-Hg2+ complex by
adjusting solution pH. Condition: 40 equiv of Hg2+ ions in water
solution containing 5 µM RMS. pH was adjusted with HClO4 and
NaOH. Inset: emission intensity ratio of RMS at 567 to 475 nm as a
function of solution pH.
concentration. An ∼100-nm emission band shift from 567 to
475 nm was achieved in the presence of 40 equiv of Hg2+ ions,
and the fluorescence intensity ratio I567/I475 changed significantly
from 11.9 to 0.4. The fluorescence color changed from orange
to cyan. Such a remarkable fluorescence color change was rarely
seen in the ever-reported ICT sensors, although a prominent
absorption wavelength shift or a weaker emission wavelength
shift was common. We would like to attribute such a significant
emission change to the coexistence of two electron-rich aniline
nitrogen atoms in the electron-donating receptor moiety, which
precluded Hg2+ ion ejection7,12 from them simultaneously in
the excited ICT fluorophore. Another attractive feature, com-
pared with sensor molecules whose emission decreased in the
presence of analytes, was the almost constant quantum yield
(0.051) of RMS at different Hg2+ ion concentrations.
FIGURE 5. 1H NMR spectra of free RMS (a) and RMS-Hg2+
complex (b) in D2O (20 equiv of Hg2+ ions were added to 5 mM RMS).
(Figure 4). The presence of other background metal ions does
not show any obvious disturbance with the signal response
induced by RMS-Hg2+ complexation (see Supporting Informa-
tion). Several factors might cooperate to achieve the unique
selectivity of RMS, such as the structural rigidity of the
o-phenylenediamine-based tetraamide receptor, the larger radius
of the Hg2+ ion, and the amide deprotonation ability of the Hg2+
ion in neat water solution.
Consistent with our early work,8 the amide arms in RMS
chelated the Hg2+ ion through an amide deprotonation process
(Figures 3 and 6), which was hampered at lower pH. This was
reflected on emission spectra by the vanishing of the peak
centered at 475 nm, assigned to the RMS-Hg2+ complex, and
the reformulation of the unbound RMS’s emission at 567 nm
with the decreasing of solution pH. A pKa1 value of 4.5 was
derived, different from that (pKa ) 1.2) of the free RMS. A
simultaneous absorption spectra shift was also observed (see
Supporting Information).
Polysulfur receptors have been extensively used1e,f in Hg2+
-
selective sensor molecules for their well-known Hg2+ affinity.
However, the O,S chelate formed by the thiazol-S and the
coumarin-carbonyl-O could not catch a Hg2+ ion in the RMS
sensor system. This is clearly seen from a CS in which the
tetraamide receptor is replaced by a tetraester one. If the O,S
chelate in the electron-withdrawing moiety caught a Hg2+ ion,
a red shift in both absorption and emission spectra should have
been observed. In fact, CS keeps silent in the presence of 40
equiv of Hg2+ ions in an acetonitrile-water solution (6:4, v/v).
The absence of Hg2+-S complexation is reasonable: (1) a sulfur
atom infused into an electron-deficient aromatic benzothiazol
ring possesses a lower Hg2+ affinity compared with that of sulfur
ether; (2) to fulfill the linear S-Hg2+-S or to realize the usual
tetrahedral14 Hg2+-ligand coordination structure may involve
an intermolecular assembly process that has a high entropic
barrier.
RMS has a notable selectivity toward the Hg2+ ion over other
heavy or transition metal ions. Only the Hg2+ ion can modulate
the fluorescence of RMS in a neutral buffered water solution
(11) A 1:1 Job’s plot does not necessarily indicate a 1:1 binding mode:
in a recently reported Pb2+ sensor molecule based on coumarin fluorophore,
a 2:2 sensor Pb2+ binding chemistry was observed, where the coumarin
carbonyl oxygen participated in sensor Pb2+ complexation. However, for
RMS, the 2:2 binding mode could be easily excluded, because metal-ion
binding at the positive pole of the coumarin fluorophore will result in a
blue-shifted spectra, while at the negative pole, it will produce a red-shifted
one; if both poles participate in complexation, the opposite effects will
counterbalance each other and result in a moderate spectra shift, instead of
the large one (60 nm) detected for the RMS-Hg2+ complex. Chen, C.-T.;
Huang, W.-P. J. Am. Chem. Soc. 2002, 124, 6246.
NMR studies of RMS (Figure 5) provide independent
evidence on Hg2+-RMS interactions. When 20 equiv of Hg2+
ions are added, the peaks, assigned to the seven aromatic
protons, experience clear (0.3-0.4 ppm) downfield shifts and
(12) If metal ion ejection takes place on an excited ICT sensor, no clear
emission spectra change could be observed.
(13) We used HEPES instead of phosphate here because some metal
ions formed precipitate in phosphate-buffered water solution.
4310 J. Org. Chem., Vol. 71, No. 11, 2006

quantum yield for the detecting of Hg2+ ions in a neutral
buffered water solution. Unfortunately, RMS-Hg2+ binding
strength, with a Ks value in the range of 104 M-1, is weak,
indicating that RMS is only effective at high Hg2+ ion
concentrations (in the present condition, Hg2+ ions could be
detected down to the micromolar range; clear emission spectra
shift was observed when Hg2+ ion concentration was down to
5 µM). Nonetheless, this work undoubtedly has paved the way
toward a highly sensitive ratiometric Hg2+ ion sensor molecule.
It is envisioned that sensor-Hg2+ binding strength will be
enhanced if the tetraamide receptor is incorporated into a “push-
pull” π-electron system, where the electron deficiency in the
electron-acceptor moiety, compared with that of RMS, is
reduced to some extent.
Experimental Section
FIGURE 6. Proposed RMS-Hg2+ complex structure and the energy-
minimized conformation by using Hyperchem software with the
molecular mechanics subroutine.17
RMS. 7 (150 mg) and 8 (90 mg, 0.408 mmol)16 were dissolved
in 15 mL of dry methanol containing five drops of piperidine. The
mixture was refluxed under nitrogen for 2.5 h. Then the solution
was cooled and concentrated under vacuum. The residue was
purified by flash chromatography using methanol-dichloromethane
(20:100, v/v) as eluant, affording 33 mg (0.046 mmol, 17%) of
RMS as a yellow solid. Mp: 195-197 °C; IR (KBr): 3368, 2937,
1655, 1615, 1549, 1056 cm-1. 1H NMR (500 MHz, D2O): δ 7.44
(s, 1 H), 6.97 (s, 1H), 6.90 (d, J ) 6.9 Hz, 3 H), 6.79 (d, J ) 7.2
Hz, 1 H), 6.44 (t, J ) 7.1 Hz, 1H), 6.36 (t, J ) 7.1 Hz, 1H), 6.20
(s, 1 H), 4.20 (s, 4 H), 3.96 (s, 4 H), 3.62-3.66 (m, 8 H), 3.31-
3.35 (m, 8 H); 13C NMR (500 MHz, D2O) δ 172.7, 172.6, 160.4,
150.9, 150.6, 149.2, 141.1, 139.0, 135.2, 126.5, 125.2, 122.9, 121.2,
113.9, 113.1, 107.1, 60.8, 55.9, 55.7, 42.3, 29.7, 14.2; HRMS (ES+)
Calcd for ([M + Na])+, 736.2377; Found, 736.2373.
get broadened. This could be attributed to a deshielding effect,
arising from the decrease of the electron density in the coumarin
fluorophore caused by N-Hg2+ (N, 6-, 7-nitrogen) complex-
ation, while the aliphatic protons, around the amide groups,
display only small downfield shifts. This could be attributed to
the shielding effect, arising from the negatively charged amide
groups (introduced by -N-Hg2+ complexation, -N-deprotonated
amide nitrogen), which, to some extent, counterbalances the
deshielding effect. In addition, ligand exchanges between the
four amide arms, on the NMR time scale, might occur since
1
only one set of aliphatic proton signals is observed. H NMR
spectra of the tetraamide receptor MR, in the presence of
different concentrations of Hg2+ ions, are also recorded (see
Supporting Information). Despite similar spectral shifting trends,
MR exhibits a higher Hg2+ ion binding strength15 since 2 equiv
of Hg2+ ions could almost bring about the maximum spectra
changes. This is reasonable in consideration of the presence of
two electron-deficient substituent groups in RMS, which
decreases the electron density on the 6-, 7-nitrogen and results
in a weaker N-Hg2+ binding strength.
CS was similarly prepared from RMS (94%) as a yellow solid.
Note: in this reaction, we used ethanol as the solvent instead of
methanol. Mp: 186-188 °C; 1H NMR (500 MHz, CDCl3): δ 8.98
(s, 1 H), 8.06 (d, J ) 8.2 Hz, 1 H), 7.95 (d, J ) 7.9 Hz, 1 H), 7.51
(t, J ) 8.1 Hz, 1 H), 7.38-7.40 (overlapped, 2 H), 6.99 (s, 1H),
4.46 (s, 4 H), 4.24 (s, 4 H), 4.10-4.19 (m, 8 H), 1.21-1.26 (m, 12
H); 13C NMR (500 MHz, CDCl3): δ 170.2, 170.1, 160.7, 160.3,
152.5, 151.5, 148.2, 141.5, 138.6, 136.6, 126.3, 125.1, 122.7, 121.9,
121.7, 117.3, 113.7, 108.0, 61.1, 60.9, 52.7, 29.7, 14.2; MS (ES+)
Calcd for ([M + H])+, 654; Found, 654.
Accordingly, on the basis of the evidence mentioned above,
Figure 6 presents a proposed Hg2+-RMS complexation struc-
ture, in which two deprotonated amide groups cooperate with
the two o-phenylenediamine nitrogen atoms to form a tetrahedral
ligand atmosphere for a Hg2+ ion. The other two unbound amide
arms may exert steric effects, which restrict the free rotation of
the amide arms and favor the Hg2+-RMS complexation.
In summary, we have developed a ratiometric ICT fluorescent
Hg2+ ion sensor RMS by incorporating an o-phenylenediamine-
derived tetraamide receptor into a coumarin platform. RMS has
several desirable sensor properties such as remarkable emission
wavelength shift, absolute selectivity, and almost constant
Acknowledgment. This work was supported by the National
Key Project for Basic Research (2003CB 114400) and the
National Natural Science Foundation of China. We thank the
reviewers for their smart and pertinent comments; some dis-
cussions originated from their comments.
Note Added after ASAP Publication. The emission intensity
ratio in Figure 3 was shown as 567 to 485 nm, instead of 475
nm, in the version published ASAP April 29, 2006; the corrected
version was published May 4, 2006.
Supporting Information Available: Synthesis and character-
ization of compounds 2-7, MR, NMR spectra, and spectrascopic
data. This material is available free of charge via the Internet at
http://pubs.acs.org.
(14) (a) Su, C.-Y.; Goforth, A. M.; Smith, M. D.; Loye, H.-C. Z. Inorg.
Chem. 2003, 42, 5685. (b) Li, G.; Song, Y.; Hou, H.; Li, L.; Fan, Y.; Zhu,
Y.; Meng, X.; Mi, L. Inorg. Chem. 2003, 42, 913.
(15) The difference in the Hg2+ ion-binding stoichiometry also occurs
1
between MR and RMS. Job’s plot using H NMR data indicates that MR
JO052642G
binds Hg2+ ion with a 1:2 stoichiometry (see Supporting Information). While
it is difficult to determine the high association constant between MR and
the Hg2+ ion using NMR techniques, a photoinduced electron transfer (PET)
fluorescent sensor molecule, based on BODIPY (boron dipyrromethene)
fluorophore and the tetraamide receptor MR, shows that the association
constant of the 1:2 MR-Hg2+ complexation is larger than 1010 M-2. This
result will be published soon in a separate paper.
(16) Compound 8 was synthesized following the literature method.
Abbotto, A.; Bradamante, S.; Facchetti, A.; Pagani, G. A. J. Org. Chem.
2002, 67, 5753.
(17) Detailed information on the calculation methods and results is
provided in Supporting Information.
J. Org. Chem, Vol. 71, No. 11, 2006 4311