Two dansyl fluorophores bearing amino acid for monitoring Hg2+ in aqueous solution and live cells

Article abstract of DOI:10.1016/j.tet.2011.03.106

A series of compounds (1-4) bearing one or two dansyl fluorophore(s) based on a Lys amino acid were synthesized in solid phase synthesis. Among them, two dansyl labeled Lys amino acid 3 detected Hg²⁺ in a 100% aqueous solution with high sensitivity (K_d=4.3 nM) via a turn-on response. Compound 3 was applied for monitoring Hg²⁺ in environmental and biological fields. 3 showed a hypersensitive response to Hg²⁺ without interferences from other metal ions and satisfied the requirements for monitoring the maximum allowable level (2 ppb) of mercury ions in drinking water demanded by EPA. In addition, 3 penetrated living HeLa cells and detected intracellular Hg²⁺. The organic spectroscopic data revealed the two sulfonamide and amide groups of 3 played a key role in stabilizing the 3-Hg ²⁺ complex.

Full text of DOI:10.1016/j.tet.2011.03.106

Tetrahedron 67 (2011) 4130e4136
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Tetrahedron
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Two dansyl fluorophores bearing amino acid for monitoring Hg2þ in aqueous
solution and live cells
*
Chuda Raj Lohani, Joung Min Kim, Keun-Hyeung Lee
Bioorganic Chemistry Lab, Department of Chemistry, Inha University, 253-Yunghyun-dong, Nam-gu, Incheon 402-751, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of compounds (1e4) bearing one or two dansyl fluorophore(s) based on a Lys amino acid were
synthesized in solid phase synthesis. Among them, two dansyl labeled Lys amino acid 3 detected Hg2þ in
a 100% aqueous solution with high sensitivity (Kd¼4.3 nM) via a turn-on response. Compound 3 was
applied for monitoring Hg2þ in environmental and biological fields. 3 showed a hypersensitive response
to Hg2þ without interferences from other metal ions and satisfied the requirements for monitoring the
maximum allowable level (2 ppb) of mercury ions in drinking water demanded by EPA. In addition,
3 penetrated living HeLa cells and detected intracellular Hg2þ. The organic spectroscopic data revealed
the two sulfonamide and amide groups of 3 played a key role in stabilizing the 3-Hg2þ complex.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 2 December 2010
Received in revised form 28 March 2011
Accepted 29 March 2011
Available online 9 April 2011
Keywords:
Hg2þ
Amino acid
Turn on
Sensor
Probe
Fluorescent
1. Introduction
fluorophore part converts the recognition events of the sensors into
fluorescent signals. Various fluorescent chemical sensors were syn-
The design and development of fluorescent chemosensors for the
detection of biologically and environmentally important metal ions
have attracted considerable interest because they allow the prompt
detection ofsmall amounts of metal ions in the field. Thedetection of
mercury ions in aqueous solutions has been of interest because
environmental contamination of highly toxic mercury ions causes
serious problems for human health and ecology.1 Various types of
fluorescent chemical sensors for Hg2þ ions have been developed.2
However, there is a need for fluorescent sensors that provide both
selective and sensitive detection of mercury ions in aqueous solu-
tions via a turn-on response because most Hg2þ sensors suffer from
at least one of the following limitations; low sensitivity in aqueous
solutions, cross sensitivity toward other heavy metal ions, turn off
response, and low solubility in aqueous solutions.2,3 Specially, as
most small chemical sensors for Hg2þ operated only in organic or
mixed aqueouseorganic solutions,4,5 they show some limitation in
practical use for monitoring Hg2þ in environmental and biological
samples. Therefore, it is challenging for developing new chemo-
sensors for Hg2þ with high sensitivity and selectivity, turn-on re-
sponse, good water solubility, and cell permeability.
thesized based on the fluorophores, such as dansyl, rhodamine, an-
thracene, naphthyl, and nile blue.6 The receptor part for specific
target metal ions was coupled with the fluorophores for the synthesis
of chemical sensors. Considering the further application of Hg2þ
chemical sensors in environmental and biological fields, the sensors
are required to have good water solubility and biological compati-
bility. As amino acids are water soluble and biologically compatible, it
is promising that development of the new Hg2þ chemical sensors
based on amino acids as a receptor part. The conjugation of fluo-
rophore into the amino acids is an important step for the synthesis of
new fluorescent chemical sensors based on amino acids. Among the
various fluorophores, the dansyl fluorophore has been used exten-
sively in a wide variety of fluorescent chemical sensors. The dansyl
fluorophore of chemical sensors has been used to detect metal ions
through chelation enhanced fluorescence (CHEF) effect and the
dansyl fluorophore is sensitive to the polarity of its microenviron-
ment via an internal charge-transfer (ICT) mechanism.6b,e,g,i,7e9
Several independent research groups including us have also
successfullysynthesized fluorescentchemicalsensors byconjugation
of dansyl fluorophore with N-terminal of peptides or amino acids.10
Accordingly, in this study, we chose a Lys residue and conjugated
one or two dansyl fluorophore(s) with the amino acid in solid phase
synthesis to synthesize a series of compounds (1e4) (Fig. 1). The
compounds were synthesized in solid phase synthesis using Fmoc-
chemistry (Supplementary data, Scheme S1).11 After cleaving the
Fluorescent chemosensors consist of a fluorophore part and a re-
ceptor part, that is, responsible for recognizing analytes.
A
* Corresponding author. E-mail address: leekh@inha.ac.kr (K.-H. Lee).
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.03.106

C.R. Lohani et al. / Tetrahedron 67 (2011) 4130e4136
4131
Fig. 1. Structures of 1, 2, 3, and 4.
product from the resin, the compounds were purified by semi-
preparative HPLC with a C18 column to produce sensors with high
purity. The successful synthesis and high purity (>98%) were con-
firmed by analytical HPLC with a C18 column and ESI mass spectrom-
eter. Solid phase synthesis and HPLC purification have the advantages
of rapid synthesis and high purity of the target compounds.
2. Result and discussion
2.1. Fluorescence emission response to metal ions
As all compounds showed good solubility in water, stock solu-
tions of the compounds were prepared in 100% distilled water and
all the photochemical experiments were carried out in a 100%
aqueous solution without a cosolvent. Fig. 2 shows the fluorescence
Fig. 3. Fluorescence emission spectra of
3
(8
m
M) in the presence of increasing
concentrations of Hg2þ (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0 equiv).
response of the compounds in the presence of each metal ion (Ca2þ
,
on calix[4]arene and rhodamine were analyzed, two or four dansyl
fluorophores were found to be necessary for the detection of heavy
and transition metal ions, such as Hg2þ, Pb2þ, or Cu2þ in aqueous
solutions or aqueouseorganic mixed solutions.9b,12,13 Upon the ad-
dition of 1 equiv of Hg2þ, approximately 15-fold enhancement of the
emission intensity at w500 nm was observed.
Cd2þ, Co2þ, Pb2þ, Cu2þ, Agþ, Mg2þ, Mn2þ, Ni2þ, Hg2þ, Zn2þ as per-
chlorate anion and Naþ, Al3þ, Kþ, as chloride anion) by excitation
with 380 nm. Compounds 1 and 4 showed no fluorescent response
to any metal ions, whereas 2 exhibits an excellent selectivity toward
Cu2þ among the metal ions tested. When 1 equiv of Cu2þ was added,
the emission intensity was considerably quenched. Interestingly, 3
exhibited outstanding selectivity toward the Hg2þ over other heavy
and transition metal ions. 3 sensitively detected only Hg2þ in 100%
aqueous solutions via turn-on response. When the structures of
fluorescent chemical sensors containing dansyl fluorophore based
2.2. Binding stoichiometry and binding affinity
The fluorescent response of 3 to the amount of Hg2þ was mea-
sured in a 10 mM HEPES buffer solution at pH 7.4. As shown in
Fig. 3, the emission intensity increased with increasing Hg2þ con-
centration. In the titration curve with Hg2þ ions, w1 equiv of Hg2þ
was required for the saturation of the emission intensity of
3 (8 mM). This suggests that although 3 has a simple structure based
on a Lys residue, it has hypersensitivity to Hg2þ ions in a 100%
aqueous solution. A Job’s plot, which exhibits a maximum at
a 0.5 mol fraction, indicates that the sensor forms a 1:1 complex
with Hg2þ (Supplementary data Fig. S1). Assuming the formation of
Fig. 2. Fluorescence spectra of (a) 1 (b) 2 (c) 3, and (d) 4 in 10 mM HEPES buffer
solution (pH¼7.4) in the presence of various metal ions (1 equiv) except Mg2þ, Ca2þ
,
Naþ, and Kþ, which were used 500 equiv (lex¼380 nm, Slit: 10/7 nm). The concen-
Fig. 4. Emission intensity change at 503 nm of 3 (10
buffer at pH 7.4 (lex¼380 nm, Slit: 10/3 nm).
m
M) with Hg2þ in 10 mM HEPES
tration of each compound is 10 mM.

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C.R. Lohani et al. / Tetrahedron 67 (2011) 4130e4136
Fig. 5. (a) Color emission changes of 3 (2
m
M) upon addition of 1 equiv of various metal ions in 10 mM HEPES buffer pH 7.4 (b) Fluorescence response of 3 (10 mM) in the presence of
Hg2þ (1 equiv) and additional various metal ions in 10 mM HEPES buffer (pH 7.4). All metal ions were evaluated at 1 equiv to Hg2þ except Naþ, Kþ, Ca2þ, and Mg2þ, which were used
at 100 equiv.
a 1:1 complex, the dissociation constant was calculated based on
the titration curve with Hg2þ by non-linear least square fit
(Supplementary data Fig. S2). The dissociation constant,
Kd (4.28ꢀ10ꢁ9 M, R2¼0.9621), indicated that 3 has tight binding
affinity to Hg2þ in a 100% aqueous solution. The sensitivity of 3 for
Hg2þ was calculated on the based on the linear relationships be-
tween the maximum emission intensity at 503 nm and the con-
centration of Hg2þ. The sensor has a detection limit of 8.7 nM
(1.7 ppb), based on 3sbi/m, where sbi is the standard deviation of
the blank measurements, and m is the slope of the intensity versus
sample concentration plot (Fig. 4). This confirms that 3 can be used
to detect qualitatively low levels of Hg2þ in aqueous solutions. The
detection limit of 3 for Hg2þ is lower than the maximum allowable
Fig. 6. Emission intensity of 3 in the presence (A) and absence of ( ) Hg2þ (1 equiv)
at different pH (lex¼380 nm).
level in drinking water (10 nM) by EPA.14 The visible emission
;
change in compound 3 was investigated in the presence and
absence of Hg2þ. As shown in Fig. 5a, 3 (2.0
m
M) showed a brighter
color in the presence of Hg2þ (2.0
mM, 1 equiv) in a 100% aqueous
excitation with 380 nm was not sensitive to pH. However, the
emission intensity of the 3-Hg2þ complex showed a dependence on
pH. 3 exhibited a sensitive turn-on response to Hg2þ with at least
10-fold enhancements in the pH range, 5.5e9.5. The largest en-
hancement was observed at pH 7.5, showing that 3 is suitable for
monitoring Hg2þ ions in physiological pH. At pH >10, the intensity
of the 3-Hg2þ complex decreased with increasing pH. This might be
due to deprotonation of the sulfonamide group pKay10 under
basic conditions. Under acidic conditions, 3 and 3-Hg2þ complex
showed very weak emission intensity. This was attributed to the
protonated dimethylamino group (pKay4) of dansyl fluorophore,
which prevents charge transfer from the dimethylamino group to
the naphthyl moiety.7e9,17
solution, whereas the color of 3 did not change in the presence of
the other metal ions tested. According to this result, the detection
limit of 3 is good enough to measure the maximum permissible
concentration of mercury in blood (1.0 mM) and urine (3.0 m
M).15
2.3. Fluorescence study in the presence of other metal ions
and at different pH
The emission spectrum of 3 in the presence of Hg2þ and other
metal ions was measured to determine the interference effect of
other metal ions on the response of 3 to Hg2þ, Fig. 5b shows the
fluorescence emission change in 3 upon the addition of each metal
ion (Ca2þ, Cd2þ, Co2þ, Pb2þ, Cu2þ, Agþ, Mg2þ, Mn2þ, Ni2þ, Hg2þ
,
Zn2þ, Naþ, Al3þ, Kþ). The Hg2þ dependent fluorescence response of
3 was unaffected by the presence of Group I and II metal ions
(5 mM), such as Naþ, Kþ, Ca2þ, and Mg2þ. In particular, the fluo-
rescence spectrum of 3-Hg2þ was not changed by other heavy and
transition metal ions (1 equiv). The change in emission spectra after
adding EDTA to the 3-Hg2þ complex that exhibited enhanced
emission intensity was investigated to examine the reversible
monitoring of Hg2þ (Supplementary data, Fig. S3). The addition of
EDTA resulted in an instant decrease in emission intensity with
110 equiv of EDTA resulting in a return to the original metal free
spectrum. EDTA is the treatment of choice to remove toxic metal
ions, Hg2þ and Pb2þ in chelation therapy.16 However, as EDTA also
bind with essential metal ions, such as Ca2þ, Fe2þ, and Cu2þ, the
long-term treatment of EDTA could induce the depletion of es-
sential minerals. Considering the more potent binding affinity than
that of EDTA for Hg2þ and water solubility of 3, it can be a potential
candidate in chelation therapy to remove Hg2þ. The effect of pH
influence on the fluorescence of 3 was examined in a 100% aqueous
solution (Fig. 6). The maximum emission intensity of free 3 by
2.4. Binding mode of 3 with Hg2D ion
To determine the binding mode of 3 with Hg2þ, the interaction
between 3 and Hg2þ was examined using organic spectroscopy
techniques, such as UV absorbance, NMR, and mass spectroscopy.
The absorption spectrum of free 3 exhibited a maximum intensity
at 216, 250, and 330 nm (Supplementary data, Fig. S4). Upon the
addition of Hg2þ, a gradual red shift in the absorbance at 330 nm
was observed and the absorbance at 380 nm increased. This sug-
gests that Hg2þ binding to 3 resulted in an increase in the conju-
gation of the naphthyl moiety of the dansyl fluorophore due to an
increase in the electron density of the naphthyl moiety.
NMR studies provided direct evidence of 3-Hg2þ interactions. As
shown in Fig. 7,1H NMR experiments were carried out in CD3CN. The
disappearance of an NH peak of the dansyl sulfonamide group at
5.7 and 6.1 ppm in the presence of Hg2þ (1 equiv) indicates that Hg2þ
coordination leads to the deprotonation of NH of the sulfonamide
group. NeHg covalent bonding formation, which is in agreement

C.R. Lohani et al. / Tetrahedron 67 (2011) 4130e4136
4133
N
13
14
12
11
15
10
O
O
HN
S
1
5
6
4
N
3
O
S
O
2
7
NH2
N
H
8
O
9
15 9 12 6
5
8
14
7
11
13
(b)
3
3
10
4
2
1
9.4
9.2
9.0
8.8
8.6
8.4
8.2
8.0
7.8
7.6
7.4
7.2
7.0
6.8
6.6
6.4
6.2
6.0
5.8
5.6
5.4
15 9
8
4
11
10
12
5
6
7
13
2
14
3
3
1
(a)
9.4
9.2
9.0
8.8
8.6
8.4
8.2
8.0
7.8
7.6
7.4
7.2
7.0
6.8
6.6
6.4
6.2
6.0
5.8
5.6
5.4
1
Fig. 7. Partial H NMR (400 MHz) of 3 (5 mM) in CD3CN at 25 ꢂC (a) in the absence and (b) presence of 1 equiv of Hg(ClO4)2.
with the common behavior of mercury cations,18 induced an in-
crease in the electron density of the naphthyl moiety of dansyl flu-
orophore, resulting in a downfield shift of the aromatic protons of
the dansyl fluorophore. The chelation of Hg2þ with the sulfonamide
groups of the dansyl fluorophores increases the electron density and
conjugation of the naphthyl moiety. This is, also supported by the
red shift in the absorbance of 3 in the presence of Hg2þ (Fig. S4).
The 3-Hg2þ complexation was also confirmed by ESI mass
spectrum (Supplementary data Fig. S5 and S6). The peak m/z
612.5579 value corresponded to [3þHþ]þ. When 1.0 equiv of Hg(II)
fluorescence response to metal ions was investigated in a 100%
aqueous solution. As shown in Fig. 2, 4 showed no fluorescent
response to any metal ions in aqueous solutions, indicating that
the amide group of 3 plays a key role in the interaction between 3
and Hg2þ. As shown in IR spectra (Supplementary data Fig. S30),
the disappearance of NH bands (3197, 2928, and 2851 cmꢁ1) of
sulfonamide and amide groups is indicative of the participation of
binding of sulfonamide and amide groups to Hg2þ. The small shift
of S]O bands from 1142 to 1112 cmꢁ1 in the presence of Hg2þ
suggests that S]O may not directly interact with Hg2þ. Based on
the organic spectroscopic data, Scheme 1 presents a proposed
3-Hg2þ complexation structure, in which the two deprotonated
sulfonamide groups cooperates with the amide group of 3 to form
a stable complex between 3 and Hg2þ. Although two dansyl moi-
eties containing sulfonamide groups are located at unsymmetrical
positions, the two sulfonamide groups and amide group of the
sensor provide sufficient interactions for the hypersensitive re-
sponse to Hg2þ in aqueous solution.
was added,
a
new peak at 810.2990 corresponding to
[3þHg2þꢁHþ]þ appeared, which indicates that 3 forms a 1:1 com-
plex with Hg2þ and a deprotonation process is induced by the
chelation of Hg2þ and 3. The amide protons display considerable
downfield shifts. This could be attributed to the shield effect,
arising from binding of amide group with Hg2þ. To confirm the
interaction between the amide group of 3 and Hg2þ, 4, which has
acid group instead of amide group, was synthesized and its
Scheme 1. Proposed binding mode of 3 with Hg2þ
.

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C.R. Lohani et al. / Tetrahedron 67 (2011) 4130e4136
Fig. 8. Bright field (a, c, e) and confocal fluorescent (b, d, f) images of HeLa cells (a, b), HeLa cells with 3 (5 mM) (c, d), and HeLa cells with 3 (5 m mM) (e, f).
M) and Hg2þ (20
2.5. Sensing mercury ions in live cells
3 equiv) in DMF (3 mL), and triethylamine (20 ml, 0.15 mmol,
3 equiv) were added. Cleavage of the peptide from the resin was
achieved by treatment with a mixture of 3 mL TFA:TIS:H2O
(95:2.5:2.5 v/v/v) at room temperature for 3 h. After filtration and
washing of the resin by TFA, a gentle stream of nitrogen was used to
remove the excess TFA. The crude was triturated with diethyl ether
chilled at ꢁ20 ꢂC and then centrifuged at 3000 rpm for 10 min
at ꢁ10 ꢂC. The crude product was purified by prep-HPLC with
a Vydac C18 column using a water (0.1% TFA)eacetonitrile (0.1% TFA)
gradient to give >97% yield. The successful synthesis was con-
firmed by ESI mass spectrometry (Platform II, micromass, Man-
chester, UK) and its homogeneity (>98%) was confirmed by reverse
phase analytical HPLC with a C18 column:
Compound 3 showed hypersensitive response to Hg2þ in a 100%
aqueous solution because it was synthesized based on Lys amino
acid, which has good water solubility. Considering the further
application in the field of biology, 3 was used to monitor Hg2þ ions
in HeLa cells to determine if the sensor can penetrate living cells
and detect intracellular Hg2þ (Fig. 8). Compound 3 (5
mM) was in-
cubated with HeLa cells for 15 min at 37 ꢂC, and then the HeLa cells
were washed with PBS. The fluorescent change in the cells was
monitored by confocal microscopy. The weak fluorescent image of
the cells indicates that 3 penetrated the cells. Significant increases
in fluorescence intensity in HeLa cells were observed upon loading
with Hg2þ (20.0
mM), demonstrating that 3 can be used to image
intracellular Hg2þ in living cells.
4.2.1. Compound 1. 1H NMR (DMSO-d6)
d
8.44 (d, 1H, J¼8.4 Hz),
8.28 (d, 1H, J¼8.4 Hz), 8.07 (d, 1H, J¼7.2 Hz), 7.98 (b, 2H), 7.88e7.85
(t, 1H, J¼6 Hz), 7.77 (s, 1H), 7.63e7.55 (m, 2), 7.51 (s, 1H), 7.25 (d, 1H,
J¼7.6 Hz), 3.61 (d, 1H, J¼5.6 Hz), 2.82 (s, 6H), 2.77e2.70 (br s, 2H),
1.60e1.56 (m, 2H), 1.37e1.34 (m, 2H), 1.26e1.22 (m, 2H); 13C NMR
3. Conclusion
A fluorescent sensor, 3 for Hg2þ was synthesized by conjugation
of two dansyl fluorophores with a Lys residue. The sensor was
developed based on amino acid, which is water soluble and
biologically compatible. The sensor selectively and sensitively
detects Hg2þ in aqueous buffer solutions via a turn-on response.
The sensor with low detection limit of 8.7 nM (1.7 ppb) can be used
to monitoring the maximum allowable level of Hg2þ in drinking
water demanded by EPA. The largest enhancement was observed at
pH 7.4, showing that 3 is suitable for monitoring Hg2þ ions in
physiological pH. The sensor penetrated live HeLa cells and detec-
ted intracellular Hg2þ. The sensor penetrated live HeLa cells and
detected intracellular Hg2þ. Therefore, it has a great potential in
practical applications for monitoring low levels of Hg2þ contami-
nation in environmental and biological samples. This information
will be useful for the further synthesis of novel chemical sensors
based on amino acids for heavy metal ions.
(DMSO-d6)
d 170.31, 151.24, 135.97, 129.31, 129.07, 129.01, 128.14,
127.82, 124.81, 123.59, 119.14, 115.16, 45.07, 42.20, 30.39, 28.81,
21.30; mp 211e213 ꢂC; ESI-MS: calcd 379.17, obsd 379.09 [MþHþ]þ.
Anal. Calcd for C18H26N4O3S: C, 57.12; H, 6.92; N, 14.80; S, 8.47.
Found: C, 57.23; H, 6.97; N, 14.87; S, 8.41.
4.2.2. Compound 2. 1H NMR (DMSO-d6)
d
8.60 (1H, J¼8.4 Hz), 8.48
(1H, J¼8 Hz), 8.30e8.26 (m, 2H), 7.83 (br s, 2H), 7.77e7.72 (m, 2H),
7.39 (d, 1H, J¼7.8 Hz), 7.35 (s,1H), 7.09 (s,1H), 3.74 (m,1H), 3.0 (s, 6H),
2.66e2.59 (m, 2H), 1.63e1.54 (m, 2H), 1.44e1.39 (m, 2H), 1.26e1.24
(m, 1H), 1.12e1.11(m, 1H); 13C NMR (DMSO-d6)
d 170.36, 151.29,
136.02, 129.36, 129.13, 129.07, 128.20, 127.87, 123.65, 119.19, 115.22,
45.12, 42.25, 30.44, 28.86, 21.35; mp 213e215 ꢂC; ESI-MS: calcd
379.17, obsd 379.09 [MþHþ]þ. Anal. Calcd for C18H26N4O3S: C, 57.12;
H, 6.92; N, 14.80; S, 8.47. Found: C, 57.26; H, 6.87; N, 14.76; S, 8.38.
4. Experimental section
4.1. General
4.2.3. Compound 3. 1H NMR (DMSO-d6)
d
8.46 (d, 1H, J¼8.4 Hz),
8.35 (d,1H, J¼8.8 Hz), 8.28e8.23 (m, 2H), 8.08e8.00 (m, 3H), 7.71 (s,
1H), 7.63 (s,1H), 7.57e7.48 (m, 3H), 7.27(d,1H, J¼7.6 Hz), 7.12 (d, 2H,
J¼6.0 Hz), 6.88 (s, 1H), 3.5 (m, 1H), 2.9 (s, 6H), 2.7 (s, 6H), 2.4 (m,
2H), 1.2 (m, 2H), 1.0 (m, 1H), 0.9 (br s, 2H), 0.6 (m, 1H); 13C NMR
Fmoc-Lys(Fmoc)eOH and Fmoc-Lys(Boc)eOH were obtained
from bead tech. Boc-Lys(Fmoc)eOH, N,N0-diisopropylcarbodiimide,
1-hydroxybenzotriazole, and Rink Amide MBHA resin were from
advanced chem tech. Other reagents for solid phase synthesis in-
cluding trifluoroacetic acid (TFA), triisopropylsilane (TIS), dansyl
chloride, triethylamine, diethyl ether, N,N0-dimethylformamide
(DMF), and piperidine were purchased from Aldrich.
(DMSO-d6)
d 173.00, 158.52, 158.18, 150.20, 150.08, 136.19, 136.09,
129.12,128.98, 128.73, 128.61, 128.44, 128.24, 127.71, 127.54, 123.81,
123.59, 120.09, 119.80, 115.52, 1155.32, 55.02, 45.18, 45.08, 41.81,
31.012, 28.10, 21.76; mp 183e184 ꢂC; ESI-MS: calcd 612.22, obsd
612.55 [MþHþ]þ. Anal. Calcd for C30H37N5O5S2: C, 58.90; H, 6.10; N,
11.45; S, 10.48. Found: C, 58.81; H, 6.16; N, 11.39; S, 10.54.
4.2. Solid phase synthesis of compounds
4.2.4. Compound 4. 1H NMR (DMSO-d6) 8.46(d, 1H, J¼8.8 Hz),
8.37e8.34 (m, 2H), 8.32e8.27 (m, 2H), 8.07(d,1H, J¼7.2 Hz), 8.03 (d,
1H, J¼3.6 Hz), 7.3 (t, 1H, J¼2.8 Hz), 7.66e7.49 (m, 4H), 7.32 (d, 1H,
J¼7.6 Hz), 7.18 (d, 1H, J¼7.6 Hz), 7.18 (d, 1H, J¼7.6 Hz), 3.41(q, 1H,
J¼7.6 Hz), 2.80 (s, 6H), 2.75 (s, 6H), 2.32e2.24 (m, 2H),1.32e1.26 (m,
Compounds were synthesized by solid phase synthesis with
Fmoc chemistry.11 Fmoc protected
L-Lys residue was assembled on
Rink Amide MBHA resin for compounds 1, 2, 3, and on 2-Cl trityl
resin for compound 4 (Supplementary data, Scheme S1). After
deprotection of Fmoc group, the coupling of dansyl chloride was
performed by the following procedure. To the resin bound amino
acid (65 mg. 0.05 mmol), dansyl chloride (40 mg, 0.15 mmol,
2H), 1.21 (s, 1H), 0.92e0.85 (m, 3H); 13C NMR (DMSO-d6)
d 173.06,
158.06, 158.23, 150.25, 150.13, 136.25, 136.15, 129.18, 129.04, 128.79,
128.67, 128.49, 128.29, 127.76, 127.59, 123.86, 123.64, 120.15, 119.86,
115.57, 115.38, 55.08, 45.24, 45.14, 41.86, 31.06, 28.16, 21.81; mp

C.R. Lohani et al. / Tetrahedron 67 (2011) 4130e4136
4135
186e187 ꢂC; ESI-MS: calcd 612.22, obsd 612.55 [MþHþ]þ. Anal.
Calcd for C30H36N4O6S2: C, 58.80; H, 5.92; N, 9.14; S, 10.47. Found: C,
58.88; H, 5.83; N, 9.22; S, 10.38.
were washed three times with PBS and confocal fluorescent
microscopy was recorded for them.
Acknowledgements
4.3. General fluorescence measurements
This work was supported by the grant (2009-0076572) from the
Basic Research Program of National Research Foundation of Korea.
The stock solution of compound 3 and 4 was prepared by dis-
solving w1 mg of 3 or 4 in 4 mL of distilled water with 5 mL (0.125%
Supplementary data
v/v) of TFA. The concentration of compound was confirmed by UV
absorbance and was diluted into 10 mM HEPES buffer solution for
the fluorescence measurement. Fluorescence emission spectrum of
a probe in a 10 mm path length quartz cuvette was measured in
10 mM HEPES buffer solution (pH 7.4) using a PerkineElmer lu-
minescence spectrophotometer (model LS 55). Emission spectra of
Experimental procedure and characterization data of com-
pounds are presented in Supplementary data. Supplementary data
related to this article can be found online at doi:10.1016/
j.tet.2011.03.106.
the probe (10 m ,
M) in the presence of various metal ions (Hg2þ, Ca2þ
References and notes
Cd2þ, Co2þ, Pb2þ, Agþ, Mg2þ, Cu2þ, Mn2þ, Ni2þ, and Zn2þ as per-
chlorate anion; and Naþ, Al3þ, and Kþ, as chloride anion) were
measured by excitation with 380 nm. The slit size for excitation and
emission was 10 and 3 nm, respectively. The concentration of the
probe was confirmed by UV absorbance at 330 nm for dansyl group.
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4.4. Determination of dissociation constant and detection
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The dissociation constant was calculated based on the titration
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related to the equilibrium concentration of the complex (HL) be-
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F ¼ F0 þ
D
F ꢀ ½HLꢃ
ꢀ
ꢁ
n
o
1=2
½HLꢃ ¼ 0:5 ꢀ KD þ LT þ HT ꢁ ð ꢁ KD ꢁ LT ꢁ HTÞ2ꢁ4 LTHT
Where F0 is the fluorescence of the probe only and
DF is the
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The detection limit was calculated based on the fluorescence
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