Near-infrared cell-permeable Hg2+-selective ratiometric fluorescent chemodosimeters and fast indicator paper for MeHG+ based on tricarbocyanines

Article abstract of DOI:10.1002/chem.201001769

Three tricarbocyanine dyes (IR-897, IR-877, and IR-925) with different thiourea substituents that function as dosimeter units through specific Hg ²⁺-induced desulfurization have been demonstrated in a fast indicator paper for Hg²⁺ an

Full text of DOI:10.1002/chem.201001769

DOI: 10.1002/chem.201001769
Near-Infrared Cell-Permeable Hg2+-Selective Ratiometric Fluorescent
Chemodosimeters and Fast Indicator Paper for MeHg+ Based on
Tricarbocyanines
Zhiqian Guo,[a] Weihong Zhu,*[a] Mingming Zhu,[b] Xumeng Wu,[a] and He Tian*[a]
Dedicated to Professor Jiacong Shen on the occasion of his 80th birthday
Abstract: Three tricarbocyanine dyes
(IR-897, IR-877, and IR-925) with dif-
ferent thiourea substituents that func-
tion as dosimeter units through specific
Hg2+-induced desulfurization have
been demonstrated in a fast indicator
paper for Hg2+ and MeHg+ ions. In
comparison with available Hg2+-selec-
tive chemodosimeters, IR-897 and IR-
877 show several advantages, such as
convenient synthesis, very long wave-
lengths falling in the near-infrared
(NIR) region (650–900 nm) with high
molar extinction coefficients, a ratio-
metric response, and quite low disturb-
ance with Ag+ and Cu2+ ions. They ex-
hibit large redshifts, which result in a
clear color change from deep blue to
pea green that can be easily monitored
by the naked eye for a convenient indi-
cator paper. In emission spectra, they
display a characteristic turn-off mode
at 780 nm and turn-on mode at 830 nm
with titration of Hg2+ ions. Remarka-
bly, the signal/noise (S/N) ratio with
other thiophilic metal ions (Ag+ and
Cu2+) is greatly enhanced with ratio-
metric measurement of two channels:
absorption point at 730 nm as excita-
tion). The distinct response is depen-
dent upon the electron-donating effect
of the thiourea substituents; that is, the
stronger the electron-donating capabili-
ty of the thiourea substituents, the
faster the Hg2+-promoted cyclization.
Additionally, experiments with living
SW1116 cells show that these three tri-
carbocyanine dyes with low toxicity
can exhibit special characteristics that
are favorable for visualizing intracellu-
lar Hg2+ and MeHg+ ions in biological
systems, including excellent membrane
permeability, minimal interfering ab-
sorption and fluorescence from biologi-
cal samples, low scattering, and deep
penetration into tissues.
excitation spectra mode (I810 nm/I670 nm
,
monitored at 830 nm) and emission
spectra mode (I830 nm/I780 nm, isosbestic
Keywords: chemodosimeters
·
fluorescence · mercury · sensors ·
tricarbocyanines
Introduction
rious is the fact that mercury ions can be converted into or-
ganomercury and accumulated, mainly as methylmercury
(MeHg+) ions, in the human body. As a strong neurotoxin,
methylmercury ions, which are mainly derivatives from the
daily diet of fish and sea mammals, affect the central nerv-
ous system to cause prenatal brain damage, various cogni-
tive and motion disorders, and Minamata disease.[2] There-
fore, considerable efforts have been made to develop selec-
tive and efficient methods[3] to monitor and perform trace
imaging of Hg2+ ions,[4] particularly fluorescence imaging of
MeHg+ ions in living cells and organisms.[5,6] Recently, cya-
nine dyes have become the focus of numerous analytical,
biological, and especially in vivo fluorescence imaging,[7] be-
cause the near-infrared (NIR) region at around 650–900 nm
exhibits special advantages including minimal interfering ab-
sorption and fluorescence from biological samples, low scat-
tering, and deep penetration into tissues.[8,9] Among cyanine
dyes, tricarbocyanines with a rigid chlorocyclohexenyl or
Mercury (Hg2+) ions, as one of the most toxic metal pollu-
tants, are widespread in air, water, and soil.[1] Even more se-
[a] Z. Q. Guo, Prof. Dr. W. H. Zhu, X. M. Wu, Prof. Dr. H. Tian
Key Laboratory for Advanced Materials and
Institute of Fine Chemicals
East China University of Science & Technology
Shanghai 200237 (P.R. China)
Fax : (+86)21-6425-2758
E-mail: whzhu@ecust.edu.cn
tianhe@ecust.edu.cn
[b] M. M. Zhu
Department of Gastroenterology, Renji Hospital
Shanghai Jiao Tong University, School of Medicine
Shanghai Institute of Digestive Disease, Shanghai 200001 (P.R.
China)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201001769.
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FULL PAPER
chlorocyclopentenyl ring in the methine chain can increase
the photostability, enhance the fluorescence quantum yield,
and provide an ideal site for further modification with
amino substitution.[10]
with Hg2+ ions, IR-877 and IR-897 exhibit large redshifts in
absorption (176 and 162 nm, respectively) to fully meet
“naked-eye” colorimetric changes, and they have been suc-
cessfully developed as Hg2+- and MeHg+-ion indicator
papers. Moreover, by monitoring either a single emission
with two excitation sources (excitation spectra mode) or
dual emission with an isosbestic absorption point as single
excitation source, these tricarbocyanines can be successfully
constructed as NIR ratiometric Hg2+ and MeHg+ chemo-
dosimeters, which allow for fast and accurate measurements
with elimination of the influence of dye concentration and
microenvironmental fluctuations in pH value, refractive
index, and photobleaching.[13b,15] The above strategy of graft-
ing the chemodosimeter unit into a tricarbocyanine provides
a fascinating approach to the design of NIR chemodosime-
ters.
Chemodosimeters have great advantages in selectivity and
sensitivity over other sensing strategies by utilization of a
specific chemical reaction (usually irreversible) between
guest molecules and target species.[11] They can perform a
chemical reaction with a specific target species (usually a
metal ion) that leads to the formation of a fluorescent or
colored product displaying a unique spectroscopic change.[12]
Indeed, several selective chemodosimeters for Hg2+ ions in
water or organic solvents have been exploited on the basis
of irreversible mercury-promoted desulfurization reactions,
including hydrolysis, cyclization, and elimination reactions
that stem from the strong thiophilic affinity of Hg2+ ions.[12]
To the best of our knowledge, however, no study has taken
these advantages for the development of a cyanine-based
NIR colorimetric and ratiometric chemodosimeter for moni-
toring Hg2+ ions or organomercury compounds. Inspired by
the chemodosimeters for Hg2+ ions and the principle of
amino-substituted tricarbocyanine dyes as NIR fluorescence
labels,[12,13b,14] we report herein three tricarbocyanine-deriva-
tive dyes (IR-897, IR-877, and IR-925; Scheme 1) with dif-
ferent thiourea substituents as Hg2+-dosimeter units through
a specific Hg2+-induced desulfurization. The electron-donat-
ing effect of the chemodosimetric groups in the three tricar-
bocyanine dyes is determined, and it is found that the stron-
ger the electron-donating capability of the thiourea substitu-
ents, the faster the Hg2+-promoted cyclization. Remarkably,
Results and Discussion
Synthetic strategy and characterization: The synthesis of the
three tricarbocyanine dyes IR-877, IR-897, and IR-925 is de-
picted in Scheme 1. In order to gain insight into the role of
the dosimetric groups for desulfurization, three thiourea de-
rivative units were obtained by the reaction of ethylenedia-
mine with isothiocyanatobenzene, N-butyl isothiocyanate,
and benzoyl isothiocyanate, respectively. Finally, the target
tricarbocyanine dyes were prepared from treatment of the
precursor chlorocyclohexenyl-substituted cyanine dye (IR-
739) with the thiourea derivatives in DMF, with a yield of
Scheme 1. Synthetic routes to IR-897, IR-877, and IR-925.
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H. Tian, W. Zhu et al.
about 30%. Their chemical structures were fully character-
1
ized by H NMR and 13C NMR spectroscopy and by HRMS
(see the Experimental Section). In an analysis of the
1H NMR spectra of the chlorocyclohexenyl-substituted tri-
carbocyanine dye IR-739 and the aminocyclohexenyl-substi-
tuted tricarbocyanine dye IR-897, the chemical shifts of the
alkene H atoms in the methine chain show significant up-
field-shift from d=6.24 and 8.48 ppm with IR-739 to d=
5.62 and 7.77 ppm with IR-897. The same tendency is also
observed with IR-877 and IR-925 (Table 1). The significant
changes in the chemical shifts of the alkene H atoms are in-
dicative of a reassignment in the electron distribution of the
tricarbocyanine dye with an intramolecular charge-transfer
(ICT) process.
Table 1. Photophysical characteristics of tricarbocyanine dyes in a mix-
ture of methanol/water (80:20).
[a]
[b]
Dye
lab
lab
Dlab
lem
e[c]
(ꢁ105)
F[d]
d (alkene H)
ACHTUNGTNER[NUNG ppm]
[nm]
[nm]
[nm]
[nm]
U
IR-739
IR-877
IR-897
IR-925
818
664
668
670
–
–
830
780
780
780
3.50
0.51
1.00
0.78
0.63
6.24, 8.48
5.60, 7.73
5.62, 7.77
5.62, 7.85
840
830
830
176
162
160
0.566
0.876
0.900
[a] Wavelength of the absorption peak after addition of Hg2+ ions.
[b] Wavelength of the emission peak. [c] Molar extinction coefficients
[mÀ1 cmÀ1] corresponding to the absorption band with no Hg2+ ion.
[d] Relative quantum yields, determined by using IR-877 (F=1.00) as a
reference.
Colorimetric detection of Hg2+ and MeHg+ ions: In com-
parison with the absorption peak of precursor dye IR-739
located at l=818 nm (Figure S1 in the Supporting Informa-
tion), IR-897 shows a very intense new band at l=668 nm
(e=8.76ꢁ104 mÀ1 cmÀ1) with
a large blueshift of about
150 nm (Table 1), which could arise from an efficient excit-
ed-state ICT process from the donor (N1, the secondary ni-
trogen atom linking with cyclohexenyl group; Scheme 2) to
Figure 1. Changes in the absorption spectra of A) IR-897 (10 mm) and
B) IR-877 (10 mm) upon titration of HgCl2 (0, 2, 4, 6, 8, 9, 10, and 12 mm)
in a mixture of methanol/water (80:20). Color changes of C) IR-897
(10 mm) and D) IR-877 (10 mm) in the presence of Hg2+ ions (10 mm) and
other metal ions (30 mm). From left to right: IR-897 or IR-877 only, with
Hg2+, Cu2+, Zn2+, Ag+, Ni2+, Cd2+, Pb2+, Co2+, and Fe2+ ions. All spec-
tra were acquired 15 min after addition of the metal ions.
Scheme 2. Mercury-promoted desulfurization reactions.
new band centered at l=830 nm (e=1.87ꢁ105 mÀ1 cmÀ1) in-
creased prominently with an isosbestic point at l=730 nm
(Figure 1A). The isosbestic point at l=730 nm clearly re-
vealed that only two species coexist; these correspond to
free IR-897 and desulfurization-product IR-863 (Scheme 2).
In the cyclization process, the central bridging secondary
amine (N1H) of the tricarbocyanine is transformed into an
imidazoline tertiary amine. This results in a decrease of elec-
tron-donating capability, so the efficiency of the excited-
the acceptor (tricarbocyanine group).[14] As expected, IR-
897 and IR-877 can be easily transformed into guanidine de-
rivatives upon titration of Hg2+ ions,[16] which results in dis-
tinct color changes in a mixture of methanol/water (80:20)
at room temperature (Figure 1A and B). With an increasing
concentration of Hg2+ ions into the IR-897 solution (10 mm),
the absorption peak at l=668 nm decreased sharply and a
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Hg2+-Selective Ratiometric Fluorescent Chemodosimeters
FULL PAPER
state ICT process from the N1
atom to the acceptor tricarbo-
cyanine was sharply diminished,
and a large redshift in the ab-
sorption spectrum with visible
color changes was thus ob-
served (Figure 1).
As a consequence, IR-897,
upon addition of Hg2+ ions, ex-
hibits an extremely large red-
shift of about 162 nm in absorp-
tion and fully meets “naked-
eye”
colorimetric
changes.
Moreover, IR-897 also displays
the same redshift from l=668
to 830 nm upon titration with
methylmercury ions (Figure 2).
Generally, colorimetric NIR
sensors with distinct color
Figure 3. HR EI mass spectra of IR-897 and IR-897 with the addition of Hg2+ ions.
moted intramolecular cyclic guanylation of the thiourea
moiety. Significant downfield shifts of the alkene protons in
the methine chain were observed from d=5.62 and
7.77 ppm to d=6.11 and 8.40 ppm (Figure 4). The original
three broad peaks at d=9.65, 9.07, and 8.34 ppm that were
assigned to the NH groups of IR-897 disappear upon addi-
tion of the Hg2+ ion, due to the intramolecular cyclic guany-
lation of the thiourea moiety. Similarly, the two groups of
CH2 peaks were downfield shifted from d=3.99 and
4.15 ppm to d=4.36 and 4.58 ppm.
Figure 2. Changes in the absorption spectra of IR-897 (10 mm) upon titra-
tion of MeHg+ (0, 1, 2, 3, and 4 equiv) in a mixture of methanol/water
(80:20). All spectra were acquired 15 min after addition of the metal
ions.
changes have the advantage of a straightforward detection
manner. As shown in Figure S2 in the Supporting Informa-
tion, visible color changes were observed when test paper
sheets adsorbed with IR-897 were dipped in MeHg+ or
Hg2+ solutions of various concentrations. Consequently, the
first fast qualitative indicator paper for MeHg+ and Hg2+
ions has been demonstrated.
Figure 4. Partial 1H NMR spectra for IR-897 showing the changes before
and after the addition of Hg2+ ions.
According to the linear Benesi–Hildebrand expression,[17]
NIR fluorescence ratiometric detection: The fluorometric
detection of Hg2+ ions with different excitation wavelengths
was also examined. As shown in Figure 5A, two different
wavelengths (l=670 and 810 nm), corresponding to excita-
tion of IR-897 and the cyclic guanylation product IR-863,
were selected as the excitation wavelengths. Upon titration
of IR-897 with Hg2+ ions, a distinct decrease was observed
in the fluorescence intensity at l=780 nm (I780 nm) for IR-
897 upon excitation at l=670 nm, until it was almost com-
pletely quenched to the baseline (Figure S4 in the Support-
ing Information). In contrast, upon excitation at l=810 nm,
the measured absorbance 1/
(AÀA0) at l=668 nm varies as a
function of 1/
ACHTUNGTRENNUNG
1:1 stoichiometry in the mercury-promoted cyclic guanyla-
tion of IR-897. The formation of 1:1 stoichiometry between
an Hg2+ ion and IR-897 was also confirmed by high-resolu-
tion ESI-MS analysis (Figure 3). The peak at m/z 770.4280
(calcd as 770.4256) corresponded to [IR-897ÀI], and the
peak at m/z 736.4382 (calcd as 736.4379) was assigned to the
1
desulfurized product [IR-863ÀI]. Moreover, H NMR stud-
ies provided further evidence consistent with the Hg2+-pro-
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H. Tian, W. Zhu et al.
This results in a fluorescence ratio based on dual emission
at l=780 and 830 nm (I830 nm/I780 nm), corresponding to the
fluorescence peaks of IR-897 and IR-863 (Figure 5B). Clear-
ly, the excitation wavelength of the isosbestic point (l=
730 nm) and the emission wavelengths (l=780 and 830 nm)
of IR-897 and IR-863 fall within the desirable NIR region;
this offers distinct advantages for both in vitro and in vivo
biological applications, in that absorption and emission
maxima in the NIR region can avoid strong interference
from UV-induced phototoxicity/autofluorescence and
sensor-induced interference encountered in the shorter
wavelength region.[7,9] Similarly, IR-877 can also be built as
a ratiometric NIR Hg2+ chemodosimeter.
The selectivity for a specific analyte over other metal ions
is an important feature of chemodosimeters. The specific se-
lectivities of IR-897 and IR-877 toward Hg2+ ions were in-
vestigated, and these analytes displayed a remarkably high
response for Hg2+ ions over other metal cations. As shown
in Figure 1, no significant changes in the color of the IR-897
solutions were observed after adding a variety of metal ions:
Cu2+, Zn2+, Ag+, Ni2+, Cd2+, Pb2+, Co2+, and Fe2+. Also,
the dependence of the fluorescence ratios at l=670 and
810 nm (I810 nm/I670 nm) upon monitoring at 830 nm with vari-
ous metal ions was measured (Figure 6A). Clearly, except
for the Hg2+ ions (I810 nm/I670 nm =7.4), the fluorescence ratios
of the various metal cations (I810 nm/I670 nm <0.25) can be ne-
glected; this includes biologically active metal ions (Na+,
Figure 5. A) Excitation spectra of IR-897 (10 mm) in the presence of Hg2+
ions at concentrations of 0, 2, 4, 6, 8, and 10 mm, monitored at l=830 nm
in a mixture of methanol/water (80:20). Inset: Emission spectra of IR-
897 (10 mm) upon titration of Hg2+ ions. lex =810 nm. B) Emission spectra
of IR-897 (1 mm) in the presence of Hg2+ ions at concentrations of 0, 0.2,
0.4, 0.6, 0.8, and 1.0 mm, excited at l=730 nm, the isosbestic point of the
absorption spectra, in a mixture of methanol/water (80:20). Inset: Fluo-
rescence intensity ratio (I780 nm/I830 nm) of IR-897 as a function of Hg2+
concentration.
K+, Ca2+, and Mg2+), trace metals in organisms (Cu2+
,
Zn2+, Ag+, Ni2+, Co2+ Mn2+, Fe3+, and Fe2+), and the prev-
alent toxic transition-metal cations (Cd2+ and Pb2+). In par-
ticular, the selectivity for Hg2+ ions over Ag+ and Cu2+ ions
becomes more distinct with the fluorescence-ratio strategy.
As a matter of fact, the signal/noise (S/N) value in the fluo-
rescence response with Hg2+ ions over Ag+ and Cu2+ ions
increases drastically from 12 and 15 times (by using the
ratio of fluorescence intensity and only monitoring at
780 nm; Figure 6B) to 30 and 40 times (by using the ratio-
metric method; Figure 6B), respectively. Similarly, in the ab-
sorption and emission spectra of IR-877, the high selectivity
for Hg2+ ions over other metal ions, including the thiophilic
Ag+ and Cu2+ metal ions, was also realized. Therefore, both
IR-877 and IR-897 exhibit high sensitivity and selectivity for
Hg2+ ions as NIR ratiometric fluorescence chemodosime-
ters.
a
“turn-on” fluorescence was observed at l=830 nm
(I830 nm); this result corresponds to the mercury-promoted
cyclic guanylation product IR-863 (Figure 5A, inset). The
detection limit of IR-897 for Hg2+ ions was evaluated at the
nanomolar level by monitoring the fluorescence titration
curves of IR-897 (10À7 m) with Hg2+ ions. Moreover, a simi-
lar fluorescence phenomenon was also observed with the ti-
tration of Hg2+ ions with IR-877.
Ratiometric measurements that involve the ratio of ab-
sorbance or emission intensity at two different wavelengths
can increase the selectivity and sensitivity to avoid trouble-
some self-calibrations and fluctuations in sensor concentra-
tion.[12c, m,13b,15] In this case, the ratiometric function can be
built with two channels. As illustrated with IR-897, with two
excitation sources at l=670 and 810 nm, corresponding to
the absorption peaks of IR-897 and the cyclic guanylation
product IR-863, we use the excitation spectra mode with
monitoring at 830 nm to obtain the ratiometric function
from the fluorescence ratio (I810 nm/I670 nm; Figure 5A). An-
other channel is to utilize a single excitation source with the
wavelength of the isosbestic absorption point at l=730 nm.
Substituent effect on response: The effect of the thiourea
substituents (Scheme 1) upon the recognition of Hg2+ ions
was further studied. IR-877 exhibits a similar photochemical
change to that of IR-897 upon addition of Hg2+ ions. As il-
lustrated in Figure 1B, the band centered at l=664 nm
shifts to l=840 nm with one isosbestic point at l=730 nm.
A large redshift (176 nm) with visible color changes from
dark blue to pea green is also observed (Figure 1B, inset).
Not only does the measuring absorbance 1/ACTHUNGTRENNUNG
664 nm vary as a function of 1/ACHTGNUTRENNUNG
ship, but also ESI MS analysis of IR-877 with Hg2+ ions
confirms a 1:1 reaction stoichiometry (Figure S3 and S8 in
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Hg2+-Selective Ratiometric Fluorescent Chemodosimeters
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Figure 6. A) Fluorescence response of IR-897 (10 mm) in the presence of
Hg2+ ions (10 mm) with various other metal ions (30 mm) in a mixture of
methanol/water (80:20). The bars represent the fluorescence intensity
ratio at l=670 and 810 nm (I810 nm/I670 nm) when monitored at 830 nm (ex-
citation spectra mode). Gray bars: each cation was added. Black bars:
each cation and Hg2+ ions were added. B) The values of signal/noise rep-
resent the changes in the absence and presence of Hg2+ ions (10 mm) with
IR-897 and IR-877 (10 mm) compared with those of Ag+ (30 mm) and
Cu2+ ions (30 mm); S: results of the method based on the fluorescence in-
tensity change at 780 nm (excited at 670 nm); R: results of the ratiomet-
ric method monitored at 830 nm (I670 nm/I810 nm) based on the excitation
mode.
Figure 7. Absorbance response of A) IR-897 (10 mm) monitored at
668 nm and B) IR-877 (10 mm) monitored at 664 nm over time with the
addition of Hg2+ ions ( : 2, : 4, : 6, : 8, 3: 10, ": 12 mm)in a mixture
~
!
&
*
of methanol/water (80:20).
sponse of IR-877 is complete within about 12 min, as deter-
mined from the absorbance changes at l=664 and 840 nm,
and emission changes at l=780 nm (Figure 7B and Fig-
ure S11 in the Supporting Information). However, establish-
ment of the equilibrium of the reaction of IR-925 (10 mm)
with Hg2+ ions (20 mm) takes over 24 h. The large difference
in the response times is related to the corresponding sub-
stituents. Compared with the butyl substituent group in IR-
877, the phenyl group in IR-897 shows a larger conjugated
system with the thiourea unit and results in more electron
richness for the sulfur atom of the thiourea unit. This
prompts the cyclization reaction of IR-897 to occur quicker
than that of IR-877. The electron-withdrawing properties of
the benzoyl group in IR-925 distinctly decrease the electron
density of the sulfur atom in the thiourea unit and make the
desulfurized cyclization of IR-925 induced with Hg2+ ions
much slower than those of IR-877 and IR-897. Consequent-
ly, the distinct responses of three tricarbocyanine dyes with
Hg2+ ions are dependent upon the electron-donating effects
of the thiourea substituents: the stronger the electron-donat-
the Supporting Information). In the ESI MS spectra, the
peak at m/z 750.4553 (calcd as 750.4569) corresponds to
[IR-877ÀI] and the peak at m/z 716.4659 (calcd as 716.4692)
is assigned to a desulfurized product (ÀH2S) [IR-843ÀI].
Unexpectedly, IR-925, which contains the electron-with-
drawing group of a benzoyl substituent, displayed almost no
response as the dosimeter unit within a reasonable time
scale upon addition of Hg2+ ions (20 mm; Figure S9 in the
Supporting Information). Even if the time period was ex-
tended to 24 h, the spectral changes of IR-925 with Hg2+
ions could be ignored with respect to those of IR-897 and
IR-877. The kinetics of the Hg2+-prompted chemodosimet-
ric cyclization was studied by analysis of the response time
towards Hg2+ ions with different thiourea substituents. The
results reveal that the cyclization reaction of IR-897 (10 mm)
with Hg2+ ion (12 mm) almost reaches completion within
5 min, as recorded by absorbance changes at l=668 and
830 nm and emission changes at l=780 nm (Figure 7A and
Figure S10 in the Supporting Information). Similarly, the re-
ing ability of the thiourea substituents, the faster the Hg2+
promoted cyclization.
-
Detection of intracellular Hg2+ ions with three tricarbocya-
nines: To further investigate the practical application of the
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H. Tian, W. Zhu et al.
three tricarbocyanine dyes in living cells, human colon
cancer cells (SW1116) were selected for our experiments.
As mentioned above, the excellent chemical and spectro-
scopic properties of the tricarbocyanine dyes with Hg2+ ions
in a mixture of methanol/water (80:20) indicate that IR-897
and IR-877 should be suitable to probe for Hg2+ ions in
living cells. Brightfield-image measurement confirms that
the cells are viable throughout the imaging studies (Fig-
ure S12a, S12d, and S12g in the Supporting Information).
After treatment of SW1116 cells with IR-897, IR-877, and
IR-925 (5 mm) alone for 20 min at 378C, the cells display
strong fluorescence (Figure S12b, S12e, and S12h in the
Supporting Information). Upon addition of Hg2+ ions
(10 mm) and incubation for another 20 min at 378C, the
SW1116 cells show a very weak fluorescence signal, which
indicates that the strong red fluorescence is almost
quenched by the cyclization reaction of IR-897 and IR-877
with intracellular Hg2+ ions (Figure S12c and S12 f in the
Supporting Information). In contrast, the cells treated with
IR-925 and incubated with Hg2+ ions exhibit an almost neg-
ligible decrease in fluorescence intensity (Figure S12i in the
Supporting Information). The results in living cells are in ac-
cordance with the investigation of the three tricarbocyanine
dyes in solution, which showed a distinct response depend-
ence upon the thiourea substituents. The above cell experi-
ments also reveal that the three tricarbocyanine dyes have
excellent membrane permeability. Thus, IR-877 and IR-897
can be expected to function as novel cell-permeable NIR
Hg2+-selective chemodosimeters in living cells by fluores-
cence microscopy.
Experimental Section
Materials and methods: All solvents were of analytical grade. The inter-
mediate of IR-739 was prepared by the established literature procedure.
1H NMR and 13C NMR spectra in CDCl3 were measured on a Brꢂcker
AV-400 spectrometer with tetramethylsilane (TMS) as an internal stan-
dard. Electrospray-ionization high-resolution mass spectra were mea-
sured on a Micromass LCT instrument under standard conditions. UV/
Vis spectra were obtained by using a Varian Cary 500 spectrophotometer
(1 cm quartz cell) at 258C. Fluorescence spectra were recorded on a
Varian Cary Eclipse fluorescence spectrophotometer (1 cm quartz cell) at
258C, and all fluorescence spectra were uncorrected by the photo-multi-
plier tube response. The width of the slit was 5 nm. Deionized water was
used to prepare all aqueous solutions. Stock solutions of Hg2+, Cu2+
,
Fe2+, Zn2+, Co2+, Ni2+, Na+, K+, Mg2+, and Ca2+ ions were prepared
from their chloride salts; solutions of Cd2+, Pb2+, and Ag+ ions were pre-
pared from their nitrate salts. All spectroscopic measurements were per-
formed in a mixture of methanol/water (80:20).
Synthesis of IR-877: IR-739 was synthesized by a general method.[18] IR-
739 (1.0 g, 1.35 mmol) and 1 (0.46 g, 2.66 mmol) were dissolved in anhy-
drous DMF (50 mL). The mixture was stirred at 80–908C for 10 h under
an argon atmosphere. The solvent was removed under reduced pressure
and then the crude product was purified by silica gel chromatography
with dichloromethane/methanol (20:1) to afford the desired product as a
1
deep blue solid (350 mg): Yield 30%; H NMR (400 MHz, CDCl3, TMS):
d=0.93 (t, J=7.2 Hz, 3H, NH
ACHTUNGTRENNUNG
NCH2CH3, NHCH2CH2CH2CH3),
NHCH2CH2CH2CH3), 1.88 (t, J=6.2 Hz, 2H, cyclohexane H), 1.99 (s,
12H, CH3), 2.55 (t, J=6.2 Hz, 4H, cyclohexane H), 3.60 (m, 2H,
NHCH2CH2CH2CH3), 3.98–4.08 (m, 8H, NHCH2CH2NH, NHCH2CH3),
5.60 (d, J=13.0 Hz, 2H, alkene H), 7.18 (d, J=8.8 Hz, 2H, Ph H), 7.37
(t, J=7.2 Hz, 2H, Ph H), 7.55 (t, J=7.2 Hz, 2H, Ph H), 7.73 (d, J=
13.0 Hz, 2H, alkene H), 7.85 (t, J=8.8 Hz, 4H, Ph H), 8.10 (d, J=8.6 Hz,
2H, Ph H), 8.63 (br, 1H, NH), 8.81 ppm (br, 1H, NH); 13C NMR
(100 MHz, CDCl3, TMS): d=11.81, 13.90, 14.13, 20.26, 21.02, 22.64,
26.36, 28.46, 31.24, 31.57, 38.21, 44.85, 49.59, 52.91, 93.15, 109.43, 119.69,
122.02, 123.78, 127.39, 128.62, 129.52, 130.02, 130.82, 131.51, 136.94,
139.89, 168.37, 169.34 ppm; HRMS (TOF-ESI+): m/z: calcd for
C49H60N5S+: 750.4569 [MÀIÀ]; found: 750.4553.
Conclusion
Synthesis of IR-897: The reaction was performed with IR-739 (1.0 g,
1.35 mmol) and 2 (0.33 g, 1.7 mmol) by using the same method as that
for IR-877. A blue solid was obtained (0.5 g): Yield 42%; 1H NMR
(400 MHz, CDCl3, TMS): d=1.40 (t, J=7.2 Hz, 6H, NCH2CH3), 1.88 (t,
J=6.2 Hz, 2H, cyclohexane H), 1.94 (s, 12H, CH3), 2.54 (t, J=6.2 Hz,
4H, cyclohexane H), 3.99 (q, J=7.2 Hz, 4H, NHCH2CH3), 4.12 (t, J=
4.8 Hz, 2H, NHCH2CH2NH), 4.15 (t, J=4.8 Hz, 2H, NHCH2CH2NH),
5.62 (d, J=13.0 Hz, 2H, alkene H), 7.12–7.18 (m, 3H, Ph H), 7.33 (t, J=
8.0 Hz, 2H, Ph H), 7.38 (t, J=8.0 Hz, 2H, Ph H), 7.52 (t, J=7.2 Hz, 2H,
Ph H), 7.77 (d, J=13.0 Hz, 2H, alkene H), 7.82–7.86 (m, 6H, Ph H), 7.98
(d, J=8.5 Hz, 2H, Ph H), 8.34 (br, 1H, NH), 9.07 (br, 1H, NH),
9.65 ppm (br, 1H, NH); 13C NMR (100 MHz, CDCl3, TMS): d=11.84,
14.10, 21.18, 22.63, 26.05, 28.48, 38.32, 44.30, 49.75, 51.74, 93.50, 109.40,
119.81, 122.12, 123.59, 123.87, 124.67, 127.42, 128.39, 128.58, 129.75,
130.00, 130.89, 131.70, 137.47, 139.30, 139.75, 168.90, 182.30 ppm; HRMS
(TOF-ESI+): m/z: calcd for C51H56N5S+: 770.4256 [MÀIÀ]; found:
770.4280.
Novel Hg2+-selective NIR fluorescent chemodosimeters
have been successfully designed by modifying an amine-sub-
stituted tricarbocyanine chromophore with dosimeter units.
The distinct response is dependent upon the electron-donat-
ing effect of the thiourea substituents and reveals that the
stronger the electron-donating ability of the thiourea sub-
stituents, the faster the Hg2+-promoted cyclization. Two ra-
tiometric strategies were performed to enhance the signal/
noise (S/N) value; these were dependent upon the two
modes of excitation spectra (I810 nm/I670 nm, monitored at
830 nm) and emission spectra (I830 nm/I780 nm, isosbestic ab-
sorption point at 730 nm as excitation). The long-wavelength
NIR-region (650–900 nm) results with the ratiometric mode
can greatly increase the selectivity and sensitivity and can
avoid the troublesome self-calibration and fluctuations in
sensor concentration. The excellent membrane permeability
and low toxicity with cells are favorable for visualizing intra-
cellular for Hg2+ and MeHg+ ions in biological systems.
Our approach can offer more convenient colorimetric and
ratiometric NIR chemodosimeters for Hg2+ and organomer-
cury ions for application in biological systems.
Synthesis of IR-925: The reaction was performed with IR-739 (1.0 g,
1.35 mmol) and 3 (0.4 g, 1.8 mmol) by using the same method as that for
IR-877.
A
blue solid was obtained (0.40 g): Yield 32%; 1H NMR
(400 MHz, CDCl3, TMS): d=1.40 (t, J=7.0 Hz, 6H, NCH2CH3), 1.86 (t,
J=6.2 Hz, 2H, cyclohexane H), 1.98 (s, 12H, CH3), 2.55 (t, J=6.2 Hz,
4H, cyclohexane H), 3.99 (m, 4H, NHCH2CH3), 4.01 (t, 2H,
NHCH2CH2NH), 4.19 (t, 2H, NHCH2CH2NH), 5.62 (d, J=13.0 Hz, 2H,
alkene H), 7.18 (t, J=8.5 Hz, 2H, Ph H), 7.37 (t, J=7.2 Hz, 2H, Ph H),
7.51–7.58 (m, 5H, Ph H), 7.82–7.88 (m, 6H, Ph H), 8.04 (d, J=8.0 Hz,
2H, Ph H), 8.38 (d, J=7.0 Hz, 2H, Ph H), 9.18 (br, 1H, NH), 9.27 ppm
(br, 1H, NH); 13C NMR (100 MHz, CDCl3, TMS): d=11.81, 14.13, 21.23,
22.65, 25.99, 28.30, 28.45, 31.58, 36.51, 38.02, 41.32, 49.70, 53.19, 93.16,
14430
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ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 14424 – 14432

Hg2+-Selective Ratiometric Fluorescent Chemodosimeters
FULL PAPER
109.39, 119.21, 122.08, 123.78, 127.36, 128.42, 128.61, 129.79, 129.96,
130.82, 131.78, 133.02, 137.18, 139.81, 168.67, 169.98 ppm.
Kim, J. Cui, X. Qian, D. R. Spring, I. Shin, J. Yoon, J. Am. Chem.
Soc. 2010, 132, 601–610.
Method for Hg2+ titration with IR-897 and IR-877: Stock solutions of
Hg2+ ions (5 mm) were prepared in deionized water. Titration experi-
ments were performed by adding a solution of IR-897 (10 mm, 3 mL) to a
quartz cell of 1 cm optical pathlength and then adding Hg2+ ions (5 mm)
incrementally by means of a micropipette. All spectra were recorded
15 min after the above operation. Test samples for selectivity experiments
were prepared by adding three equivalents of the appropriate other
metal ion to a solution of IR-897 (10 mm, 3 mL). All test solutions were
shaken for 1 min and then kept at room temperature for 15 min. The ex-
periment of Hg2+-ion titration with IR-877 was carried out by a similar
procedure to the described above.
[6] a) M. Santra, D. Ryu, A. Chatterjee, S. K. Ko, I. Shin, K. H. Ahn,
Chem. Commun. 2009, 2115–2117; b) Y. K. Yang, S. K. Ko, I. Shin,
J. Tae, Org. Biomol. Chem. 2009, 7, 4590–4593; c) H. Strasdeit,
Angew. Chem. 2008, 120, 840–842; Angew. Chem. Int. Ed. 2008, 47,
828–830; d) X. Chen, K. H. Baek, Y. Kim, S. J. Kim, I. Shin, J.
Yoon, Tetrahedron 2010, 66, 4016–4021.
[7] a) C. Bouteiller, G. Clave, A. Bernardin, B. Chipon, M. Massonneau,
P. Y. Renard, A. Romieu, Bioconjugate Chem. 2007, 18, 1303–1317;
b) M. J. Yuan, Y. L. Li, J. B. Li, C. H. Li, X. F. Liu, J. Lv, J. L. Xu,
H. B. Liu, S. Wang, D. B. Zhu, Org. Lett. 2007, 9, 2313–2316; c) R.
Guliyev, A. Coskun, E. U. Akkaya, J. Am. Chem. Soc. 2009, 131,
9007–9013; d) G. T. Dempsey, M. Bates, W. E. Kowtoniuk, D. R.
Liu, R. Y. Tsien, X. Zhuang, J. Am. Chem. Soc. 2009, 131, 18192–
18193; e) U. Mayerhçffer, K. Deing, K. Gruß, H. Braunschweig, K.
Meerholz, F. Wꢂrthner, Angew. Chem. 2009, 121, 8934–8937;
Angew. Chem. Int. Ed. 2009, 48, 8776–8779; f) D. Oushiki, H.
Kojima, T. Terai, M. Arita, K. Hanaoka, Y. Urano, T. Nagano, J.
Am. Chem. Soc. 2010, 132, 2795–2801; g) X. Li, W. Shi, S. Chen, J.
Jia, H. Ma, O. S. Wolfbeis, Chem. Commun. 2010, 2560–2562; h) N.
Ramsay, A. Jemth, A. Brown, N. Crampton, P. Dear, P. Holliger, J.
Am. Chem. Soc. 2010, 132, 5096–5014.
Cell-culture methods: The cell line SW1116 was provided by Shanghai
Institute of Digestive Disease, China. The cell line SW1116 in this study
was cultured in Rosewell Park Memorial Institute 1640 (RPMI-1640)
medium (Gibco, USA) supplemented with 10% fetal bovine serum and
grown at 378C in a humidified 5% CO2 atmosphere. Cells were seeded
onto 12-well plates at an initial density of 1ꢁ105 cells per well and al-
lowed to adhere for 24 h.
Fluorescence imaging: After the SW1116 cells had been washed with
phosphate-buffered saline (PBS), 5 mm tricarbocyanine dye in PBS was
added to the cells. The cells were then incubated for 20 min at 258C. Cell
imaging was carried out after washing of the cells with PBS. The cells
were further treated with HgCl2 solution (10 mm) in PBS for 20 min. The
treated cells were imaged by fluorescence microscopy (OLYMPUS
BX51, Japan, with a 20ꢁ objective lens).
[8] a) W. Leevy, S. T. Gammon, H. Jiang, J. R. Johnson, D. J. Maxwell,
E. N. Jackson, M. Marquez, D. Worms, B. D. Smith, J. Am. Chem.
Soc. 2006, 128, 16476–16477; b) Z. R. Zhang, S. Achilefu, Org. Lett.
2004, 6, 2067–2070; c) C. Li, T. R. Greenwood, Z. M. Bhujwalla, K.
Glunde, Org. Lett. 2006, 8, 3623–3626.
[9] J. V. Frangioni, Curr. Opin. Chem. Biol. 2003, 7, 626–634.
[10] a) L. Strekowski, M. Lipowska, G. Patonay, J. Org. Chem. 1992, 57,
4578–4580; b) H. Lee, J. C. Mason, S. Achilefu, J. Org. Chem. 2006,
71, 7862–7865.
[11] a) V. Dujols, F. Ford, A. W. Czarnik, J. Am. Chem. Soc. 1997, 119,
7386–7387; b) J. Kovꢅcs, T. Rçdler, A. Mokhir, Angew. Chem. 2006,
118, 7979–7981; Angew. Chem. Int. Ed. 2006, 45, 7815–7817; c) R.
Martꢃnez-MꢅÇez, F. Sancenꢄn, Coord. Chem. Rev. 2006, 250, 3081–
3093; d) F. Song, S. Watanabe, P. E. Floreancig, K. Koide, J. Am.
Chem. Soc. 2008, 130, 16460–16461.
Acknowledgements
This work was financially supported by the NSFC/China, the National
Basic Research 973 Program (2006CB806200), the Program for Professor
of Special Appointment at Shanghai Institutions of Higher Learning, the
Shanghai Shuguang Project (07SG34), the Specialized Research Fund for
the Doctoral Program of Higher Education (SRFDP 200802510011), and
the State Key Laboratory of Precision Spectroscopy (ECNU).
[12] a) M.-Y. Chae, A. W. Czarnik, J. Am. Chem. Soc. 1992, 114, 9704–
9705; b) G. Hennrich, H. Sonnenschein, U. Resch-Genger, J. Am.
Chem. Soc. 1999, 121, 5073–5074; c) B. Liu, H. Tian, Chem.
Commun. 2005, 3156–3158; d) Y. K. Yang, K.-J. Yook, J. Tae, J. Am.
Chem. Soc. 2005, 127, 16760–16761; e) J. Ros-Lis, M. D. Marcos, R.
Martinez-Manez, K. Rurack, J. Soto, Angew. Chem. 2005, 117,
4479–4482; Angew. Chem. Int. Ed. 2005, 44, 4405–4407; f) S. K. Ko,
Y. K. Yang, J. Tae, I. Shin, J. Am. Chem. Soc. 2006, 128, 14150–
14155; g) K. C. Song, J. S. Kim, S. M. Park, K. C. Chung, S. Ahn, S.-
K. Chang, Org. Lett. 2006, 8, 3413–3416; h) J. S. Wu, I. C. Hwang,
K. S. Kim, J. S. Kim, Org. Lett. 2007, 9, 907–910; i) M. H. Lee, B. K.
Cho, J. Yoon, J. S. Kim, Org. Lett. 2007, 9, 4515–4518; j) X. Chen,
S. W. Nam, M. J. Jou, Y. Kim, S. J. Kim, S. Park, J. Yoon, Org. Lett.
2008, 10, 5235–5238; k) M. Yu, M. Shi, Z. Chen, F. Li, X. Li, Y.
Cao, J. Xu, H. Yang, Z. Zhou, T. Yi, C. Huang, Chem. Eur. J. 2008,
14, 6892–6900; l) H. Zheng, G. Q. Shang, S. Y. Yang, X. Gao, J. G.
Xu, Org. Lett. 2008, 10, 2357–2360; m) X. Zhang, Y. Xiao, X. Qian,
Angew. Chem. 2008, 120, 8145–8149; Angew. Chem. Int. Ed. 2008,
47, 8025–8029; n) M. H. Lee, S. W. Lee, S. H. Kim, C. Kang, J. S.
Kim, Org. Lett. 2009, 11, 2101–2104.
[13] a) E. Sasaki, H. Kojima, H. Nishimatsu, Y. Urano, K. Kikuchi, Y.
Hirata, T. Nagano, J. Am. Chem. Soc. 2005, 127, 3684–3685; b) K.
Kiyose, H. Kojima, Y. Urano, T. Nagano, J. Am. Chem. Soc. 2006,
128, 6548–6549; c) B. Tang, H. Huang, K. Xu, L. Tong, G. Yang, X.
Liu, L. An, Chem. Commun. 2006, 3609–3611; d) S. Tatay, P. GaviÇ,
E. Coronado, E. Palomares, Org. Lett. 2006, 8, 3857–3860; e) B.
Tang, L. J. Cui, K. H. Xu, L. L. Tong, G. W. Yang, L. G. An, Chem-
BioChem 2008, 9, 1159–1164; f) M. Zhu, M. Yuan, X. Liu, J. Xu, J.
Lv, C. Huang, H. Liu, Y. Li, S. Wang, D. Zhu, Org. Lett. 2008, 10,
1481–1484; g) R. Shunmugam, G. J. Gabriel, C. E. Smith, K. A.
Aamer, G. N. Tew, Chem. Eur. J. 2008, 14, 3904–3907; h) B. Tang, F.
[1] a) G. Guzzi, C. A. M. La Porta, Toxicology 2008, 244, 1–12; b) N.
Basu, A. Scheuhammer, N. Grochowina, K. Klenavic, D. Evans, M.
O Brien, M. Chan, Environ. Sci. Technol. 2005, 39, 3585–3591.
[2] a) T. W. Clarkson, L. Magos, G. J. Myers, N. Engl. J. Med. 2003, 349,
1731–1739; b) C. M. L. Carvalho, E.-H. Chew, S. I. Hashemy, J. Lu,
A. Holmgren, J. Biol. Chem. 2008, 283, 11913–11923.
[3] a) J. P. Desvergne, A. W. Czarnik, Chemosensors of Ion and Mole-
cule Recognition, Springer, New York, 1997; b) A. P. de Silva,
H. Q. N. Gunaratne, T. Gunnlaugsson, A. J. M. Huxley, C. P. McCoy,
J. T. Rademacher, T. E. Rice, Chem. Rev. 1997, 97, 1515–1566; c) B.
Valeur, I. Leray, Coord. Chem. Rev. 2000, 205, 3–40; d) R. Martꢃ-
nez-MaÇez, F. Sancenꢄn, Chem. Rev. 2003, 103, 4419–4476; e) J. S.
Kim, D. T. Quang, Chem. Rev. 2007, 107, 3780–3799.
[4] a) E. M. Nolan, S. J. Lippard, Chem. Rev. 2008, 108, 3343–3480, and
references therein; b) S. Yoon, A. E. Albers, A. P. Wong, C. J.
Chang, J. Am. Chem. Soc. 2005, 127, 16030–16031; c) A. Coskun,
E. U. Akkaya, J. Am. Chem. Soc. 2006, 128, 14474–14475; d) S. V.
Wegner, A. Okesli, P. Chen, C. He, J. Am. Chem. Soc. 2007, 129,
3474–3475; e) Z. Q. Guo, W. H. Zhu, L. J. Shen, H. Tian, Angew.
Chem. 2007, 119, 5645–5649; Angew. Chem. Int. Ed. 2007, 46, 5549–
5553; f) D. Wu, A. B. Descalzo, F. Weik, F. Emmerling, Z. Shen,
X. Z. You, K. Rurack, Angew. Chem. 2008, 120, 199–203; Angew.
Chem. Int. Ed. 2008, 47, 193–197; g) L. Liu, D. Q. Zhang, X. P.
Zheng, Z. Wang, D. B. Zhu, J. Nanosci. Nanotechnol. 2009, 9, 3975–
3981; h) G. Mor-Piperberg, R. Tel-Vered, J. Elbaz, I. Willner, J. Am.
Chem. Soc. 2010, 132, 6878–6879.
[5] a) F. Qian, C. Zhang, Y. Zhang, W. He, X. Gao, P. Hu, Z. Guo, J.
Am. Chem. Soc. 2009, 131, 1460–1468; b) Z. Xu, K.-H. Baek, H. N.
Chem. Eur. J. 2010, 16, 14424 – 14432
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
14431

H. Tian, W. Zhu et al.
Yu, P. Li, L. Tong, X. Duan, T. Xie, X. Wang, J. Am. Chem. Soc.
2009, 131, 3016–3023.
2003, 5, 4465–4468; d) S. Dahmenand, S. Brꢆse, Org. Lett. 2000, 2,
3563–3565.
[14] X. Peng, F. Song, E. Lu, Y. Wang, W. Zhou, J. Fan, Y. Gao, J. Am.
Chem. Soc. 2005, 127, 4170–4171.
[17] a) H. A. Benesi, J. H. Hildebrand, J. Am. Chem. Soc. 1949, 71,
2703–2707; b) M. Barra, C. Bohne, J. C. Scaiano, J. Am. Chem. Soc.
1990, 112, 8075–8579.
[15] a) M. A. Haidekker, T. P. Brady, D. Lichlyter, E. A. Theodorakis, J.
Am. Chem. Soc. 2006, 128, 398–399; b) D. Srikun, E. Miller, D. W.
Domaille, C. J. Chang, J. Am. Chem. Soc. 2008, 130, 4596–4597.
[16] a) J. Manimala, E. Anslyn, Eur. J. Org. Chem. 2002, 3909–3922;
b) R. A. Batey, D. A. Powell, Org. Lett. 2000, 2, 3237–3240; c) D.
Boeglin, S. Cantel, A. Heitz, J. Martinez, J. A. Fehrentz, Org. Lett.
[18] N. Narayanan, G. Patonay, J. Org. Chem. 1995, 60, 2391–2395.
Received: June 23, 2010
Published online: October 29, 2010
14432
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Chem. Eur. J. 2010, 16, 14424 – 14432