Thiourea-based fluorescent chemosensors for aqueous metal ion detection and cellular imaging

Article abstract of DOI:10.1021/jo500710g

We describe three significant advances in the use of thioureas as reporting elements for metal-responsive fluorescent chemosensors. First, on the basis of the crystal structure of a chemosensor analogue, we provide a deeper understanding of the details of the thiourea coordination environment. Second, we describe a new generation of chemosensors with higher affinities for Zn ²⁺ and Cd²⁺ than were observed for earlier probes, expanding the scope of this type of probe beyond Hg²⁺ detection. Third, we show that a thiourea-based chemosensor can be employed for fluorescence microscopy imaging of Hg²⁺ ion concentrations in living mammalian cells.

Full text of DOI:10.1021/jo500710g

Article
pubs.acs.org/joc
Thiourea-Based Fluorescent Chemosensors for Aqueous Metal Ion
Detection and Cellular Imaging
Mireille Vonlanthen,† Colleen M. Connelly,‡ Alexander Deiters,‡,§ Anthony Linden,†
and Nathaniel S. Finney*,†,‡
†Department of Chemistry, University of Zurich, Zurich CH-8057, Switzerland
‡Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695, United States
§Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, United States
S
* Supporting Information
ABSTRACT: We describe three significant advances in the
use of thioureas as reporting elements for metal-responsive
fluorescent chemosensors. First, on the basis of the crystal
structure of a chemosensor analogue, we provide a deeper
understanding of the details of the thiourea coordination
environment. Second, we describe a new generation of
chemosensors with higher affinities for Zn2+ and Cd2+ than
were observed for earlier probes, expanding the scope of this
type of probe beyond Hg2+ detection. Third, we show that a
thiourea-based chemosensor can be employed for fluorescence microscopy imaging of Hg2+ ion concentrations in living
mammalian cells.
In an effort to address these issues, we have worked to
develop sulfur-based functional groups as reporting elements
and have recently reported the use of sulfoxides and thioureas
to this end.5,6 Building on our first generation of thiourea−
naphthalimide conjugates,6 we describe here a second
generation of PET-based thiourea probes with high affinities
for metal ions in aqueous media.7,8 We find that the new
chemosensors are insensitive to excess acid and can be applied
in cellular imaging.
INTRODUCTION
■
As an analytical technique, fluorescence is remarkable for
combining great sensitivity with ease of measurement. A central
liability is that few analytes of interest are intrinsically
fluorescent. This discrepancy has driven the development of
fluorescent chemosensors, molecules that convert reversible
association with a nonfluorescent analyte into a fluorescence
response.1 Fluorescent chemosensor development has been
particularly effective for metallic species, and selective probes
for several metal ions of biological and environmental relevance
(e.g., Ca2+, Zn2+, and Hg2+) are now available.2−4
The dominant approach to turning reversible metal binding
into a fluorescence response is the disruption of the electronic
interaction between a nitrogen atom lone pair and a proximal
fluorophore via metal coordination by nitrogen.1 Most
common are the use of benzylic amine−fluorophore con-
jugates, in which photoinduced electron transfer (PET) from
nitrogen quenches the fluorescence, and anilinic fluorophores,
in which the nitrogen lone pair is engaged in the formation of
an intramolecular charge transfer (ICT) excited state. In the
former case, protonation or coordination suppresses the
quenching and leads to enhanced emission. In the latter,
protonation or coordination leads primarily to variation in the
fluorophore emission wavelength, although concomitant
intensity variation is not uncommon.
RESULTS AND DISCUSSION
■
First-Generation Thiourea Probes. Our validation of
thioureas as PET quenchers in metal-responsive fluorescent
chemosensors has so far relied on naphthalimide chromophores
because of their synthetic accessibility, chemical robustness, and
visible emission.9 We have found that the fluorescent
chemosensors MePic and DiPic (Figure 1) have low intrinsic
quantum yields as a result of PET quenching of the
fluorescence emission by the thioureas (Figure 2).6 Emission
from the pendant naphthalimide reporter can be recovered by
metal ion coordination of the thiourea (Table 1) with up to 20-
fold fluorescence enhancement. The addition of a large excess
of H+ (TFA) did not produce any change in the fluorescence
emission of MePic or DiPic, validating our hypothesis that, in
contrast to the amine-based probes described above, thioureas
would provide H+-independent signaling. Of the range of metal
ions screened, the most significant fluorescence enhancements
While these nitrogen-centric motifs have proven to be quite
general,1 reliance on nitrogen coordination for signaling
possesses two characteristic shortcomings: false-positive signal-
ing resulting from protonation (i.e., pH sensitivity) and an
exclusive reliance on nitrogen coordination chemistry.
Received: March 26, 2014
Published: June 12, 2014
© 2014 American Chemical Society
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Table 1. Values of (I/I0)max and ϕmax for Titrations of MePic
a
and DiPic
sensor
Zn2+
Cd2+
Hg2+
Pb2+
b
Titrations in CH3OH
21.7 (0.65)
MePic
DiPic
20.5 (0.62)
13.0 (0.65)
19.0 (0.57)
7.4 (0.37)
6.3 (0.19)
c
5.0 (0.25)
−
d
Titrations in H2O
c
MePic
DiPic
9.3 (0.28)
5.7 (0.29)
4.6 (0.14)
4.5 (0.23)
4.4 (0.13)
8.6 (0.43)
−
−
Figure 1. Fluorescent chemosensors MePic and DiPic (Py = 2-
c
pyridyl).
a
ϕmax in parentheses. ϕmax = ϕ0 × (I/I0)max, where ϕ0(MePic) = 0.03
b
and ϕ0(DiPic) = 0.05. Titrations in CH3OH at 3.3 μM chemosensor.
c
d
were observed for the d10 cations Zn2+, Cd2+, and Hg2+,
although MePic provides a modest response to Pb2+ as well. Of
these, Hg2+ was by far the most tightly bound (Table 2), and
the DiPic·Hg2+ complex was found to form with Kd ≤ 15 nM in
99:1 H2O/CH3CN.6,10
No detectable fluorescence response. Titrations in 9:1 H2O/
CH3OH at 3.3 μM chemosensor.
Table 2. Apparent log(Kd/M) for Titrations of MePic and
DiPic
While the metal ion affinities of MePic and DiPic are similar,
there is significant variation in the maximum quantum yield,
which reflects details of the thiourea coordination geometry
that are not yet fully understood. We have obtained a crystal
structure of the 1:1 complex of ZnCl2 and a truncated analogue
of DiPic in which the fluorophore-bearing side chain is replaced
by a phenyl group (Figure 3).11 The structure reveals a near-
planar thiourea and coordination of the metal center by a single
pyridine unit. Notably, the coordinated zinc atom is located
significantly out of the thiourea plane, with an N−C−S−Zn
dihedral angle of 57.40(14)°. Bis(thiourea)zinc chloride is
known to have zinc coordinated in the plane of the thiourea,12
consistent with σ coordination by an sp2-hybridized S atom
presumably the intrinsically preferred geometry. The observed
deviation from in-plane coordination must be a consequence of
geometric constraints imposed by the ring formation required
for coordination of the picolyl group. From a functional point
of view, these observations indicate that the coordination
geometry responds strongly to structural details of the chelating
group, which foreshadows the modulation of metal ion affinity
and fluorescence response by ligand variation.
sensor
Zn2+
Cd2+
Hg2+
Pb2+
a
Titrations in CH3OH
MePic
DiPic
−3.2
−2.9
−3.2
−3.1
Titrations in H2O
−5.8
−6.1
−2.5
b
−
c
b
MePic
DiPic
−1.1
−0.9
−2.1
−2.3
−5.9
−6.0
−
d
b
−
a
b
Titrations in CH3OH at 3.3 μM chemosensor. No detectable
fluorescence response. Titrations in 9:1 H2O/CH3OH at 3.3 μM
chemosensor. Lower-concentration titrations revealed that this
log(Kd/M) value is at least −7.8. See ref 6.
c
d
Consistent with the crystal structure, all of the solution-phase
titrations of MePic and DiPic indicated 1:1 ligand:metal
stoichiometry.6 The Hg2+ affinity is sufficient to warrant
evaluation of these probes in environmental samples, and it
will be shown that DiPic is suitable for use in cellular imaging
(vide infra).
A limitation of these first-generation fluorescent chemo-
sensors is that the nitrogen atoms of the thiourea remain
conjugated to and coplanar with the thiocarbonyl. As a result,
the thiourea nitrogen centers are not available for coordination
and only one of the distal N-substituents can participate in
cooperative metal binding with the thiourea, as can be seen in
the crystal structure (Figure 3). This accounts for the relatively
low affinity observed for Zn2+ despite the presence of the high-
Zn2+-affinity dipicolyl fragment in DiPic and for the similarities
Figure 3. Molecular structure of the 1:1 complex between ZnCl2 and a
truncated analogue of DiPic.
in the metal affinities of MePic and DiPic. In conjunction with
the induced deviation from optimal thiourea coordination
discussed above, overcoming this limitation clearly requires
Figure 2. Fluorescent chemosensing with MePic and DiPic.
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more extended ligand domains with a greater degree of
flexibility in order to accommodate alternate coordination
geometries.
Second-Generation Thiourea Probes. In order to
increase the flexibility and coordination denticity of the
metal-binding domains, analogues with ethylenediamine (En)
spacers, EnMePic and EnDiPic, were prepared. In these
molecules, the picolyl-bearing nitrogen is no longer conjugated
to the thiocarbonyl, removing the associated geometric
constraint and freeing the lone pair for binding (Figure 4).
aqueous titrations, the magnitude of the fluorescence response,
represented by (I/I0)max, is generally lower than in CH3OH,
and the responses to Ag+ and Pb2+ are negated, as indicated by
the fact that the log(Kd/M) values were too high (> −1) to be
accurately determined. No fluorescence enhancement for
EnMePic or EnDiPic is observed at pH as low as 5.5.17 The
response of EnMePic in aqueous solution is diminished relative
to MePic (e.g., there is no response to aqueous Cd2+). In
contrast, the enhancements seen with EnDiPic are comparable
or superior to those for DiPic and are uniformly great enough
(I/I0 > 5) to be potentially useful (Table 3). EnMePic and
EnDiPic retain their high affinities for Hg2+ in 9:1 H2O/
CH3OH, but EnDiPic now has pronounced affinities for Zn2+
and Cd2+ as well (Table 4), exceeding the affinity for Hg2+. The
log(Kd/M) values determined from the titrations are at the
limit of measurement for the current reporting fluorophore,
which is not bright enough to allow data acquisition below ∼1
μM without repeat measurements. As a result, we think that the
actual affinities are greater than the determined values, although
the relative affinities are likely correct. For reasons discussed
below, we have not pursued lower-concentration measure-
ments. The stoichiometries of the Hg2+ and Cd2+ complexes are
1:1, as determined by Job plots and/or Hill coefficients.18,19
While EnMePic·Zn2+ is also a 1:1 complex, EnDiPic·Zn2+ has
2:1 L:M stoichiometry.
There is not a clear correlation between the metal ion affinity
and the magnitude of the fluorescence response. This is
consistent with a complexation model in which the affinity is
determined by the ethylenediamine fragment and the
fluorescence response reflects the details of a second intra-
molecular equilibrium involving coordination of the thiourea
(Figure 5). We have previously observed that thioureas have
low intrinsic affinities for metal ions, allowing us to exclude
direct thiourea coordination as a significant process.6
The affinities and intensity of fluorescence response of
EnDiPic would appear to set the stage for applied measure-
ments. As it turns out, although the compounds are entirely
stable on the time scale of our titrations,20 we found that
EnMePic and EnDiPic degrade over the course of a day in
solution and 1−2 weeks as isolated solids. The degradation
leads to the formation of complex mixtures of polar material
and appears to involve intramolecular cyclization coupled with
Figure 4. EnMePic and EnDiPic.
Mono-Boc-protected ethylenediamine was either dialkylated to
form the dipicolyl derivative 1 or monoalkylated by reductive
amination to give 3 (Scheme 1).13 In the latter case, N-
methylation provided the requisite protected diamine.14
Following removal of the Boc group, the free amines
(RNH2) 2 and 4 were reacted with the common imidazolyl
thiourea 5, described previously,6 to afford EnDiPic and
EnMePic, respectively, in modest yields.15
Metal Ion Titrations. With the new probes in hand, metal
ion titrations were performed in CH3OH with Na+, K+, Mg2+,
Ca2+, Ag+, Zn2+, Cd2+, Hg2+, and Pb2+. Metal ion solutions were
prepared from the corresponding metal chlorides, nitrates, or
perchlorates. CH3OH (as opposed to aqueous) solutions were
chosen for initial evaluation to allow comparison with previous
data and to identify binding events too weak to be observed
with H2O as the competing solvent.
Metal ion response was observed for Ag+, the d10 metal
cations, and Pb2+ but not for alkaline or alkaline-earth cations.
Ag+, Zn2+, Cd2+, Hg2+, and Pb2+ titrations were then repeated in
9:1 H2O/CH3OH (Tables 3 and 4).11,16 Focusing on the
Scheme 1. Preparation of EnMePic and EnDiPic
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a
Table 3. Values of (I/I0)max and ϕmax for Titrations of EnMePic and EnDiPic
sensor
Zn2+
Cd2+
Titrations in CH3OH
Hg2+
Ag+
Pb2+
b
d
EnMePic
EnDiPic
7.4 (0.22)
7.4 (0.30)
5.1 (0.15)
3.2 (0.13)
6.3 (0.19)
9.0 (0.36)
5.2 (0.16)
2.4 (0.10)
−
1.6 (0.06)
c
Titrations in H2O
d
d
d
EnMePic
EnDiPic
1.9 (0.06)
−
2.5 (0.08)
7.4 (0.30)
−
−
−
−
d
d
11.2 (0.45)
5.3 (0.21)
a
b
ϕmax in parentheses. ϕmax = ϕ0 × (I/I0)max, where ϕ0(EnDiPic) = 0.04 and ϕ0(EnMePic) = 0.03. Titrations in CH3OH at 3.3 μM chemosensor.
Titrations in 9:1 H2O/CH3OH at 3.3 μM chemosensor. No detectable fluorescence response.
c
d
Table 4. Apparent log(Kd/M) for Titrations of EnMePic and
EnDiPic
sensor
Zn2+
Cd2+
Hg2+
Ag+
Pb2+
a
Titrations in CH3OH
c
EnMePic
EnDiPic
−5.0
−7.1
−6.9
−7.0
−5.9
−6.0
−5.6
−5.7
−
−5.4
b
Titrations in H2O
c
c
c
EnMePic
EnDiPic
−2.4
−6.9
−
−5.9
−6.1
−
−
c
c
−7.2
−
−
a
b
Titrations in CH3OH at 3.3 μM chemosensor. Titrations in 9:1
H2O/CH3OH at 3.3 μM chemosensor. No detectable fluorescence
c
response.
dealkylation to form cyclic guanidine species. This conclusion is
based on the high polarity and MS analysis of the crude
degraded material; we have not yet been able to isolate purified
degradation products.21
Figure 6. Fluorescence images of HeLa cells treated with DiPic and
HgCl2. (a) HeLa cells treated with a DMSO control overnight at 37
°C. (b) Cells treated with a DMSO control overnight followed by
HgCl2 (200 μM) for 10 min at 37 °C. (c) Cells incubated with DiPic
(20 μM) overnight at 37 °C. (d) Cells treated with DiPic (20 μM)
overnight followed by HgCl2 (200 μM) for 10 min at 37 °C. Imaging
was performed on a Zeiss Axio Observer inverted microscope using a
63× objective and excitation and emission wavelengths of 365 nm and
445/50 nm, respectively.
Cellular Assays. We were unable to deploy EnDiPic in
cellular assays because of its instability on the assay time scale.
However, we have evaluated DiPic as a probe for Hg2+ in living
human cells, as Hg2+ is a logical starting point on the basis of
the high affinity and selectivity of DiPic for this ion. We found
that DiPic is sufficiently membrane-permeant to enter HeLa
cells and that it allows visualization of the presence of Hg2+ in a
concentration-dependent manner.11 HeLa cells were incubated
with DiPic (20 μM) overnight in standard DMEM growth
medium, treated with HgCl2 (200 μM) for 10 min, and
examined by fluorescence imaging. In the absence of either
DiPic or Hg2+ ions, no detectable fluorescence was observed,
similar to that of control cells treated with DMSO only (Figure
6a−c). However, upon treatment with both DiPic and HgCl2, a
significant increase in fluorescence appeared throughout the
cells but predominantly in the cytoplasm (Figure 6d). This
demonstrates that the sensor DiPic is capable of detecting Hg2+
in live human cells. Moreover, DiPic successfully detected Hg2+
ions at lower concentrations of 50 and 100 μM, and the
fluorescence intensity showed Hg2+ concentration depend-
ence.11
based thiourea probes in live cells, providing a solid precedent
for the design and evaluation of future thiourea probes for
relevant trace thiophilic ions (e.g., Fe2+ or Cr3+).22
Next-Generation Thiourea Probes. Two important
issues must be addressed in the next generation of thiourea
probes: enhancement of the stability and brightness of the
reporting fluorophore. The need to improve the stability is self-
evident; we expect that, as observed with the first-generation
fluorophores,6 N-methylation of the thiourea will improve the
stability. The use of more absorptive fluorophores will allow
lower-concentration measurements.
CONCLUSIONS
■
We have shown that thiourea−naphthalimide conjugates with
extended binding domains function as metal-responsive
fluorescent chemosensors in aqueous media. They are capable
While the practical value of imaging Hg2+ in biological
systems may be limited,4 these data validate the use of PET-
Figure 5. Proposed model for metal ion coordination (Fl = fluorophore; M = metal cation).
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of providing a useful (I/I0 > 5) turn-on response to Zn2+, Cd2+,
and Hg2+ with at least high-nanomolar sensitivity. In parallel, a
previously reported thiourea has been shown to be a viable
optical imaging agent for Hg2+ in live cells. Sufficient structural
understanding has been attained to allow the design of the next
generation of improved thiourea-based fluorescent chemo-
sensors, which should exhibit enhanced stability and improved
measurement sensitivity and be suitable for environmental and
further cellular applications.
temperature and stirred for 2 h, and then 2 N NaOH(aq) was added.
The aqueous phase extracted with CH2Cl2, and the combined organic
fractions were dried over MgSO4 and concentrated. 2 was obtained
without purification as a yellow oil (48 mg, 85%). The spectroscopic
data were consistent with those previously reported.13
1H NMR (δ ppm, 400 MHz, CDCl3): 8.54 (dd, 2H, J = 4.9, J = 1.3),
7.65 (td, 2H, J = 7.7, J = 1.8), 7.48 (d, 2H, J = 7.8), 7.15 (ddd, 2H, J =
7.4, J = 4.9, J = 1.2), 3.86 (s, 4H), 2.82 (t, 2H, J = 6.0), 2.69 (t, 2H, J =
6.0). 13C NMR (δ ppm, 100 MHz, CDCl3): 159.5 (2), 149.1 (2),
136.5 (2), 123.0 (2), 122.1 (2), 60.5 (2), 56.3, 39.3. HRMS-ESI: calcd
for C14H19N4 [M + H]+ 243.16020, found 243.16045.
EXPERIMENTAL SECTION
4-(2-Methoxyethoxy)-N-(ethyl-1-(2-(bis(pyridin-2-ylmethyl)-
amino)ethyl) thiourea)naphthalimide (EnDiPic). 5 (53 mg, 0.12
mmol), acetonitrile (5 mL), and 2 (30 mg, 0.12 mmol) were
combined in CH3CN (5 mL), and the mixture was stirred for 2 h at
reflux. After removal of the solvent, the crude product was purified by
column chromatography (Al2O3; CH2Cl2 → CH2Cl2/MeOH, 99:1) to
give the product EnDiPic (45 mg, 60%) as a yellow solid.
■
General Notes. Imidazolyl thiourea 5, DiPic, and MePic were
prepared as previously described.6 All of the other reagents were used
as received. Synthetic procedures were carried out under an inert
atmosphere in dry solvent using standard Schlenk techniques, unless
otherwise noted. Flash chromatographic purification was performed
using silica gel Merck 60 (particle size 0.040−0.063 mm) packed in
glass columns; the eluting solvent for each purification was determined
by thin-layer chromatography (TLC). Analytical TLC was performed
using Merck TLC silica gel 60 F254 or Macherey−Nagel POLY-
GRAM ALOX N/UV254.
1H NMR (δ ppm, 400 MHz, CDCl3): 8.64 (dd, 1H, J = 1.6, J = 8.4),
8.59 (dd, 1H, J = 1.2, J = 7.3), 8.57−8.56 (m, 2H), 8.54 (d, 1H, J =
8.3), 7.71 (dd, 1H, J = 7.3, J = 8.4), 7.59 (dt, 2H, J = 1.8, J = 7.6),
7.35−7.33 (m, 2H), 7.12−7.09 (m, 2H), 7.04 (d, 1H, J = 8.3), 4.50−
4.48 (m, 2H), 4.45−4.43 (m, 2H), 3.96−3.93 (m, 4H), 3.85 (s, 4H),
3.53 (s, 3H), 2.82 (t, 2H, J = 5.6). 13C NMR (δ ppm, 100 MHz,
CDCl3): 181.9, 165.4, 164.8, 160.6, 159.0 (2), 149.3 (2), 136.7 (2),
134.0, 132.1, 129.7, 129.5, 126.2, 123.8, 123.3 (2), 122.3 (2), 122.1,
114.9, 106.3, 70.8, 68.7, 59.9, 59.6, 52.3, 43.0, 39.2. HRMS-ESI: calcd
for C32H34N6O4S [M + H]+ 599.24350, found 599.24344; calcd for
C32H33N6NaO4S [M + Na]+ 621.22545, found 621.22522. Rf (Al2O3;
CH2Cl2/MeOH, 99.5:0.5): 0.25.
N-(2-Picolyl)-N′-Boc-ethylenediamine (3). N-Boc-1,2-diamino-
ethane (627 mg, 3.92 mmol) was dissolved in methanol (15 mL),
and pyridine-2-carboxaldehyde (350 mg, 3.27 mmol) was added at 0
°C, and the reaction mixture was stirred at room temperature for 3 h.
Sodium triacetoxyborohydride (970 mg, 4.57 mmol) was added, and
the reaction mixture was stirred overnight. The solvent was removed
under vacuum, and the residue was treated with saturated Na2CO3.
The product was extracted with CH2Cl2, and the organic phase was
dried over MgSO4 and concentrated under vacuum. 3 was obtained as
a brown oil (680 mg, 83%) in sufficient purity for use in the
subsequent step. The spectroscopic data were consistent with those
previously reported.14
1H NMR chemical shifts are reported in parts per million relative to
the solvent residual peak (CDCl3, 7.26 ppm). Multiplicities are given
as s (singlet), d (doublet), t (triplet), q (quartet), dd (doublet of
doublets), or m (multiplet), and the coupling constants (J) are given
in hertz. 13C NMR chemical shifts are reported relative to the solvent
residual peak (CDCl3, 77.0 ppm). HRMS data were acquired on a
UHPLC-HR-MS QTOF instrument with an ESI source. All of the
synthetic products were noncrystalline (oils or sticky solids),
precluding melting point determinations. Fluorescence measurements
were carried out in spectroscopic grade CH3OH using 450 W xenon
lamp excitation with 1 nm excitation and 1 nm emission slit widths.
Emission spectra were obtained by excitation at the longest-
wavelength absorption maxima. For extinction coefficient determi-
nations, four independent solutions with different concentrations were
prepared, with absorption between 0.04 and 0.10 AU. The values of ε
were calculated by linear least-squares fitting of plots of A versus
concentration. All of the fits gave R2 values of ≥0.98. The quantum
yields for EnMePic and EnDiPic were determined by standard
methods11,23 using anthracene (ϕ = 0.30) in CH3OH.24 The samples
were diluted to optical transparency (A ≤ 0.05), and the integrated
emission intensity was compared to that of an isoabsorptive solution of
the standard in degassed solvent. Metal ion titrations were performed
as previously described using metal solutions prepared with
spectroscopic grade CH3OH or unbuffered, purified H2O.6
Synthetic Procedures. N,N-Bis(2-picolyl)-N′-Boc-ethylenedi-
amine (1). 2-Chloromethylpyridine hydrochloride (451 mg, 2.75
mmol) and Na2CO3 (530 mg, 5.00 mmol) were added to a solution of
N-Boc-1,2-diaminoethane (200 mg, 1.25 mmol) in methanol (20 mL),
and the reaction mixture was heated to reflux for 48 h. A 2 N solution
of NaOH (20 mL) was added, and the product was extracted with
dichloromethane. The organic phase was washed with brine, dried
over MgSO4, and concentrated under vacuum. The crude product was
purified by column chromatography (Al2O3; eluent: hexanes/CH2Cl2,
1:1 → CH2Cl2/MeOH, 99:1) to give the product as a brown-yellow
oil (220 mg, 52%). The spectroscopic data were consistent with those
previously reported.13
1H NMR (δ ppm, 400 MHz, CDCl3): 8.56 (dd, 1H, J = 5.0, J = 1.2),
7.65 (td, 1H, J = 7.7, J = 1.8), 7.29 (d, 1H, J = 7.8 Hz), 7.18 (ddd, 1H,
J = 7.5, J = 4.9, J = 1.1), 5.17 (s, 1H), 3.93 (s, 2H), 3.39−3.36 (m, 2H),
2.82−2.80 (m, 2H), 1.44 (s, 9H). 13C NMR (δ ppm, 125 MHz,
CDCl3): 158.9, 156.2, 149.4, 136.6, 122.4, 122.2, 79.2, 54.5, 48.8, 40.2,
28.4 (3). Rf (Al2O3; CH2Cl2/MeOH, 99.5:0.5): 0.11.
N-Methyl-N-(2-picolyl)-N′-Boc-ethylenediamine (Boc-Protected
4). Iodomethane (34 mg, 0.239 mmol) and sodium carbonate (85
mg, 0.80 mmol) were added to a solution of 3 (50 mg, 0.20 mmol) in
CH3OH (20 mL), and the reaction mixture was heated to reflux for 72
h. The solvent was concentrated under vacuum, and the crude product
was purified by column chromatography (Al2O3; eluent: CH2Cl2). The
product was obtained as a light-yellow oil (25 mg, 48%).
1H NMR (δ ppm, 400 MHz, CDCl3): 8.57−8.55 (m, 1H), 7.67 (td,
1H, J = 7.7, J = 1.8), 7.38 (d, 1H, J = 7.9), 7.18 (dd, 1H, J = 7.4, J =
3.0), 3.71 (s, 2H), 3.27−3.25 (m, 2H), 2.59−2.57 (m, 2H), 2.32 (s,
3H), 1.45 (s, 9H). 13C NMR (δ ppm, 125 MHz, CDCl3): 159.0, 156.2,
149.3, 136.6, 123.2, 122.2, 79.2, 63.8, 56.5, 42.4, 38.2, 38.6 (3).
HRMS-ESI: calcd for C14H24N3O2 [M + H]+ 266.1863, found
266.1864. Rf (Al2O3; CH2Cl2): 0.31.
N-Methyl-N-(2-picolyl)ethylenediamine (4). N-Methyl-N-(2-picol-
yl)-N′-Boc-ethylenediamine (150 mg, 0.57 mmol) was cooled to 0 °C
in CH2Cl2 (10 mL), and trifluoroacetic acid (1.61 g, 14.15 mmol) was
added. The mixture was allowed to warm to room temperature and
stirred for 2 h. Then 2 N NaOH(aq) was added, and the aqueous
phase was extracted with CH2Cl2. The combined organic fractions
were dried over MgSO4 and concentrated. 4 was obtained without
1H NMR (δ ppm, 400 MHz, CDCl3): 8.55 (ddd, 2H, J = 4.9, J =
1.8, J = 0.9), 7.64 (td, 2H, J = 7.6, J = 1.8), 7.43 (d, 2H, J = 7.8), 7.16
(ddd, 2H, J = 7.5, J = 4.9, J = 1.2), 5.80 (s, 1H), 3.89 (s, 4H), 3.25−
3.22 (m, 2H), 2.75−2.72 (m, 2H), 1.44 (s, 9H). 13C NMR (δ ppm,
125 MHz, CDCl3): 159.2 (2), 156.2, 149.1 (2), 136.5 (2), 123.2 (2),
122.1 (2), 78.8, 60.2 (2), 53.5, 38.5, 28.5 (3). HRMS-ESI: calcd for
C19H27N4O2 [M + H]+ 343.21333, found 343.21285; calcd for
C19H26N4NaO2 [M + Na]+ 365.19510, found 365.19480. Rf (CH2Cl2/
MeOH, 99:1): 0.2.
N,N-Bis(2-picolyl)ethylenediamine (2). 1 (80 mg, 0.23 mmol) was
cooled to 0 °C in CH2Cl2 (2 mL), and trifluoroacetic acid (660 mg,
5.85 mmol) was added. The mixture was allowed to warm to room
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purification as a yellow oil (48 mg, 50%) and used immediately in the
next reaction.
Fluorescence Sensing; Springer Science: London, 2009; Chapter 6.
(g) Tsukanov, A. V.; Dubanosov, A. D.; Bren, V. A.; Minkin, V. I.
Chem. Heterocycl. Compd. 2008, 44, 899−923. (h) Callan, J. F.; de
Silva, A. P.; Magri, D. C. Tetrahedron 2005, 61, 8551−8588. (i) 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,
1515−1566.
1H NMR (δ ppm, 400 MHz, CDCl3): 8.57−8.55 (m, 1H), 7.68−
7.64 (m, 1H), 7.38 (d, 1H, J = 7.9), 7.19−7.17 (m, 1H), 3.68 (s, 2H),
2.56−2.54 (m, 4H), 2.26 (s, 3H).
4-(2-Methoxyethoxy)-N-(ethyl-1-(2-methyl(2-picolyl)amino)-
ethyl)thiourea)naphthalimide (EnMePic). 5 (103 mg, 0.24 mmol),
acetonitrile (5 mL), and 4 (40 mg, 0.24 mmol) were combined in
CH3CN (5 mL), and the mixture was stirred for 2 h at reflux. The
solvent was removed, and the crude product was purified by column
chromatography (Al2O3; CH2Cl2 → CH2Cl2/MeOH, 99:1) to give the
product EnMePic as a yellow solid (45 mg, 36%).
(2) For an overview of fluorescent probes for Ca2+, see: Simpson, A.
W. M. Methods Mol. Biol. 2013, 937, 3−36.
(3) For an overview of fluorescent Zn2+ detection, see: Huang, Z.;
Lippard, S. J. Methods Enzymol. 2012, 505, 445−468.
(4) For overviews of fluorescent Hg2+ detection, see: (a) Kim, H. N.;
Ren, W. X.; Kim, J. S.; Yoon, J. Chem. Soc. Rev. 2012, 41, 3210−3244.
(b) Nolan, E. M.; Lippard, S. J. Chem. Rev. 2008, 108, 3443−3480.
(5) Kathayat, R. S.; Finney, N. S. J. Am. Chem. Soc. 2013, 135,
12612−12614.
1H NMR (δ ppm, 400 MHz, CDCl3): 8.66−8.55 (m, 3H), 8.53 (d,
1H, J = 8.3), 7.70 (dd, 1H, J = 7.3, J = 8.4), 7.62 (td, 1H, J = 1.8, J =
7.7), 7.34 (d, 1H, J = 7.8), 7.14 (dt, J = 2.9, J = 8.1), 7.05 (d, 1H, J =
8.3), 4.57−4.36 (m, 4H), 4.01−3.81 (m, 4H), 3.73 (s, 2H), 3.52 (s,
5H), 2.71 (s, 2H), 2.31 (s, 3H). 13C NMR (δ ppm, 125 MHz, CDCl3):
182.0, 165.4, 164.7, 160.6 (2), 149.4, 136.8, 134.0, 132.1, 129.6, 129.6,
126.2, 123.7, 123.4, 122.4, 122.0, 114.8, 106.0, 70.8, 68.6, 63.3, 59.6,
55.4, 53.6, 42.2, 39.1. HRMS-ESI: calcd for C27H32N5O4S [M + H]+
522.21695, found 522.21711. Rf (Al2O3; CH2Cl2/MeOH, 99.5:0.5):
0.25.
(6) Vonlanthen, M.; Finney, N. S. J. Org. Chem. 2013, 78, 3980−
3988.
(7) For reviews of anion coordination by ureas/thioureas, see:
(a) Gunnlaugsson, T.; Ali, H. D. P.; Glynn, M.; Kruger, P. E.; Hussey,
G. M.; Pfeffer, F. M.; Santos, C. M. G.; Tierney, J. J. Fluoresc. 2005, 15,
287−299. (b) Amendola, V.; Bonizzoni, M.; Esteban-Gomez, D.;
Fabbrizzi, L.; Licchelli, M.; Sancenon, F.; Taglietti, A. Coord. Chem.
Rev. 2006, 250, 1451−1470. (c) Gunnlaugsson, T.; Glynn, M.; Tocci,
G. M.; Kruger, P. E.; Pfeffer, F. M. Coord. Chem. Rev. 2006, 250,
3094−3117. (d) Li, A.-F.; Wang, J.-H.; Wang, F.; Jiang, Y.-B. Chem.
Soc. Rev. 2010, 39, 3729−3745. (e) Duke, R. M.; Veale, E. B.; Pfeffer,
F. M.; Kruger, P. E.; Gunnlaugsson, T. Chem. Soc. Rev. 2010, 39,
3936−3953.
Cell Culture. Experiments were performed using HeLa cells
(ATCC) cultured in Dulbecco’s Modified Eagle Medium (DMEM)
(Hyclone) supplemented with 10% fetal bovine serum (FBS)
(Hyclone) and 2% penicillin/streptomycin (Mediatech) and main-
tained at 37 °C in a 5% CO2 atmosphere.
Detection of Hg2+ Ions in Mammalian Cells. HeLa cells were
passaged into an eight-well chamber slide at 10 000 cells/well and
grown overnight at 37 °C. The medium was removed, and the cells
were treated with either a DMSO control (1% DMSO final
concentration) or DiPic (20 μM) in DMEM and then incubated
overnight at 37 °C. The medium was removed, and the cells were
washed three times with PBS (100 μL). The cells were then treated
with HgCl2 at 0 or 200 μM in PBS and incubated for 10 min at 37 °C.
The cells were imaged for fluorescence on a Zeiss Axio Observer
inverted microscope using a 63× objective and filter set 49 (excitation
365 nm, emission 445/50 nm).
(8) For an early example of a PET-based chemosensor that does not
rely on nitrogen, see: de Silva, A. P.; Sandanayake, K. R. A. S. J. Chem.
Soc., Chem. Commun. 1989, 1183−1185. For other examples, see
especially ref 1i.
(9) For overviews of the versatility of naphthalimide fluorophores,
see ref 7e and: Banerjee, S.; Veale, E. B.; Phelan, C. M.; Murphy, S. A.;
Tocci, G. M.; Gillespie, L. J.; Frimannsson, D. O.; Kelly, J. M.;
Gunnlaugsson, T. Chem. Soc. Rev. 2013, 42, 1601−1618.
(10) For reports of Hg2+-responsive fluorescent chemosensors based
on aminomethylanthracene thioureas, in which PET has been claimed
as the operant signaling mechanism, see: (a) Profatilova, I. A.; Bumber,
A. A.; Tolpygin, I. E.; Rybalkin, V. P.; Gribanova, T. N.; Mikhailov, I.
E.; Bren, V. A. Russ. J. Gen. Chem. 2005, 75, 1774−1781. (b) Tolpygin,
I. E.; Shepelenko, E. N.; Revinskii, Y. V.; Tsukanov, A. V.; Dubonosov,
A. D.; Bren, V. A.; Minkin, V. I. Russ. J. Gen. Chem. 2010, 80, 756−770.
(11) See the Supporting Information.
ASSOCIATED CONTENT
■
S
* Supporting Information
Representative absorbance, emission, and titration spectra;
concentration-dependent Hg2+ imaging in HeLa cells; ORTEP
for the structure in Figure 3; copies of H and 13C NMR
1
spectra; and crystallographic data for the structure in Figure 3
(CIF). This material is available free of charge via the Internet
at http://pubs.acs.org.
(12) Kunchur, N. R.; Truter, M. R. J. Chem. Soc. 1958, 3478−3484.
(13) N,N-Bis(picolyl)ethylenediamine: (a) Romary, J. K.; Barger, J.
D.; Bunds, J. E. Inorg. Chem. 1968, 7, 1142−1145. By reductive
amination: (b) Matouzenko, G. S.; Bousseksou, A.; Lecocq, S.; van
Koningsbruggen, P. J.; Perrin, M.; Kahn, O.; Collet, A. Inorg. Chem.
1997, 36, 2975−2981. In Boc-protected form: (c) Hanaoka, K.;
Kikuchi, K.; Urano, Y.; Nagano, T. J. Chem. Soc., Perkin Trans. 2 2001,
1840−1843.
AUTHOR INFORMATION
Corresponding Author
*E-mail: nsfinney@ncsu.edu.
■
Notes
The authors declare no competing financial interest.
(14) Lim, N. C.; Ewart, C. B.; Bowen, M. L.; Ferreira, C. L.; Barta, C.
A.; Adam, M. J.; Orvig, C. Inorg. Chem. 2008, 47, 1337−1345.
ACKNOWLEDGMENTS
abs
(15) EnMePic: ε = 13 800 cm−1 M−1; ϕ = 0.03; λ = 368 nm
■
max
em
max
This work was supported by the Swiss National Science
Foundation, the Institute of Organic Chemistry (UZH), and a
GlaxoSmithKline graduate fellowship (C.M.C.).
(longest-wavelength maximum); λ = 446 nm. EnDiPic: ε = 14 500
abs
max
cm−1 M−1; ϕ = 0.04; λ = 368 nm (longest-wavelength maximum);
em
λ
= 446 nm.
max
(16) Binding constants were determined by nonlinear least-squares
fitting of plots of emission intensity vs log[M] using the program
Prism3 (Graphpad, Inc., San Diego, CA).
(17) The addition of HCl to a solution of EnMePic or EnDiPic does
not produce a change in fluorescence until pH ∼5. At this point, the
protonated picolylamine begins to serve as an alternate PET acceptor,
and the fluorescence begins to increase. For precedent, see: de Silva, S.
A.; Zavaleta, A.; Baron, D. E.; Allam, O.; Isidor, E. V.; Kashimura, N.;
Percarpio, J. M. Tetrahedron Lett. 1997, 38, 2237−2240.
REFERENCES
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