New fluorescent and colorimetric chemosensors based on the rhodamine and boronic acid groups for the detection of Hg2+

Article abstract of DOI:10.1016/j.tetlet.2010.04.067

Novel rhodamine B (RB) derivatives bearing mono and bis-boronic acid groups were investigated as Hg²⁺ selective fluorescent and colorimetric sensors. These derivatives are first examples of reversible fluorescent chemosensors for Hg²⁺

Full text of DOI:10.1016/j.tetlet.2010.04.067

Tetrahedron Letters 51 (2010) 3286–3289
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
New fluorescent and colorimetric chemosensors based on the rhodamine
and boronic acid groups for the detection of Hg2+
Sook Kyung Kim a, , K. M. K. Swamy a,b, , So-Young Chung a, Ha Na Kim a, Min Jung Kim a, Yongsuk Jeong c,
Juyoung Yoon a,c,
*
a Department of Chemistry and Nano Science BK21, Ewha Womans University, Seoul 120-750, Republic of Korea
b Department of Pharmaceutical Chemistry, V. L. College of Pharmacy, Raichur 584 103, India
c Department of Bioinspired Science, Ewha Womans University, Seoul 120-750, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Novel rhodamine B (RB) derivatives bearing mono and bis-boronic acid groups were investigated as Hg2+
selective fluorescent and colorimetric sensors. These derivatives are first examples of reversible fluores-
cent chemosensors for Hg2+ which utilized boronic acid groups as binding sites. Two new RB-boronic acid
derivatives displayed selective ‘Off–On’-type fluorescent enhancements and distinct color changes with
Hg2+. Selective fluorescent enhancement of two rhodamine derivatives was attributed to ring opening
from the spirolactam (nonfluorescent) to ring-opened amide (fluorescent).
Article history:
Received 25 February 2010
Revised 9 April 2010
Accepted 16 April 2010
Available online 20 April 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Sensors based on the ion-induced changes in fluorescence have
been actively investigated due to the simplicity and high detection
limit of the fluorescence.1 Inorganic mercury is one of the most
prevalent toxic metals in the environment, and gains access to
the body orally or dermally. When absorbed in the human body,
these species result in damage to the brain, kidneys, and endocrine
system.2 Due to the high toxicity of mercury, considerable atten-
tion has been devoted to the development of new fluorescent
chemosensors for the detection of mercury and mercuric salts with
sufficient selectivity.3,4
For the syntheses of rhodamine-boronic acid derivatives, first of
all, rhodamine ethylamine 4 was prepared following the reported
procedure.4g Intermediate
4 was then reacted with 2-form-
ylphenylboronic acid in ethanol, which was refluxed for 6 h. The
imine intermediate was further reacted with NaBH4 to give 110
(41%) as pink solid after the silica column chromatography using
CH2Cl2–MeOH (95:5, v/v) as eluent (Scheme 1). On the other hand,
bis-boronic acid derivative 211 was prepared in 37% yield by treat-
Boronic acids are known to have high affinities for substances
that contain vicinal diol groups (e.g., carbohydrates) and, conse-
quently, they have been used in novel boronic acid-based fluores-
cent chemosensors for carbohydrates.5 Recently, boronic acid
derivatives have been also utilized as sensors for fluoride6 or cya-
nide ions.7 However, only one example is available for the use of
boronic acid-based fluorescent chemosensor to detect metal ion
(Cu2+) directly.8
Herein, we report two new rhodamine derivatives bearing
mono and bis-boronic acid groups (1 and 2) as selective fluorescent
and colorimetric sensors for Hg2+, in which spirolactam (nonfluo-
rescent) to ring-opened amide (fluorescent) process9 was utilized
for the detection of Hg2+. Even though various rhodamine deriva-
tives have been reported as fluorescent chemosensors or chemod-
osimeters,9 no precedents existed for the use of boronic acid-based
rhodamine to detect Hg2+ directly.
(HO)2B
B(OH)2
O
O
NH2
N
H
1)
NH
N
N
N
O
2) NaBH4
N
O
4
N
O
1
B(OH)2
Br
Br
(HO)2B
O
N
N
O
(HO)2B
N
N
N
N
O
2
N
O
N
* Corresponding author. Tel.: +82 2 3277 2400; fax: +82 2 3277 2384.
E-mail address: jyoon@ewha.ac.kr (J. Yoon).
3
Contributed equally to this work.
Scheme 1. Synthesis of compounds 1–3.
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.04.067

S. K. Kim et al. / Tetrahedron Letters 51 (2010) 3286–3289
3287
ment of 4 with 2-bromomethylphenylboronic acid followed by sil-
120
100
80
60
40
20
0
ica column chromatography using CH2Cl2–MeOH (96:4, v/v) as elu-
ent (Scheme 1). The control compound 312 was prepared following
the similar procedure using benzyl bromide in 54% yield. These
new compounds were fully characterized by NMR and high resolu-
tion FAB mass spectroscopy. The 1H NMR, 13C NMR spectra are ex-
plained in Supplementary data.
2+
Hg
The perchlorate salts of Ag+, Ca2+, Cd2+, Co2+, Cs+, Cu2+, Hg2+, K+,
Li+, Mg2+, Mn2+, Na+, Ni2+, Sr2+, and Zn2+ ions were used to evaluate
+
+
+
2 only, Na , K , Cs
2+ 2+ 2+
2+
2+
the metal ion binding properties of compounds 1 and 2 (3 lM) in
Zn , Sr , Cd , Co
2+ 2+ 2+
2+
Cu
CH3CN–water (9:1, v/v). The fluorescence spectra were obtained
by excitation of the rhodamine fluorophore at 510 nm. Among
these metal ions (50 equiv), both of these probes showed large
fluorescence enhancements only with Hg2+ (Figs. 1 and 2) even
though there were relatively smaller fluorescence enhancements
with Cu2+. Upon the addition of Hg2+ to a colorless solution of com-
pound 1 or 2, both a red color and the fluorescence characteristics
of rhodamine B appear. Due to the weakening of both upon the
addition of excess EDTA (Supplementary Fig. 9) to the solution of
2 and Hg2+, it is believed that this process is reversible. The large
fluorescence enhancement as well as the colorimetric change can
be attributed to the spirolactam ring opening, which was induced
by the complexation of Hg2+. The time-dependent fluorescence re-
Ca , Ni , Mn , Mg
2+
Pb
520
540
560
580
600
620
640
Wavelength (nm)
Figure 2. Fluorescent changes of 2 (3
CH3CN–DDW (9:1, v/v) (kex = 510 nm, Slit: 3 nm/3 nm).
l
M) with various metal ions (150
l
M) in
sponses of probe 2 (3 l
M) to Hg2+ (50 equiv) were monitored at
580 nm under pseudo-first-order kinetic conditions (Supplemen-
tary Fig. 8). Under these conditions, the observed rate constants
(kobs) at CH3CN were found to be 0.01733 minÀ1 for Hg2+. The fluo-
rescence emissions were obtained after 30 min after the addition
of metal ions.
Figures 3 and 4 explain the fluorescent titrations of 1 and 2 with
Hg2+ in CH3CN–water (9:1, v/v). The association constants of 1 and
2 with Hg2+ were calculated as 3.3 Â 103 MÀ1 and 2.1 Â 104 MÀ1
(error <15%).13 The 1:1 stoichiometry was also confirmed by the
Job plots as shown in the Supplementary data. Bis-boronic probe
2 displayed as high as ninefold tighter binding with Hg2+ compared
to mono-boronic probe 1, which can be attributed to an additional
boronic acid ligand in the case of 2. On the other hand, compound
3, which does not contain any boronic acid group, did not show
large fluorescent changes with metal ions even though there was
a very small enhancement with Cu2+ (Fig. 5). This observation
along with larger binding constant of 2 suggests that boronic acid
moiety plays an important role in the binding with Hg2+. The car-
bonyl oxygen as well as oxygens of boronic acid groups can make a
Figure 3. Fluorescent titrations of 1 (3
(kex = 510 nm, Slit: 3 nm/5 nm).
l
M) with Hg2+ in CH3CN–H2O (9:1, v/v)
nice binding pocket for the Hg2+
.
For probe 2, the fluorescent changes were examined in 100%
CH3CN and CH3CN–HEPES buffer (pH 7.4, 0.01 M) (9:1, v/v) too.
120
2+
Hg
100
80
60
40
20
0
2+ 2+
2+
1only, Zn , Ni , Co ,
2+
2+
2+
+
2+
Sr , Cd , Ca , Mg
,
Figure 4. Fluorescent titrations of 2 (3
(kex = 510 nm, Slit: 3 nm/3 nm).
l
M) with Hg2+ in CH3CN–H2O (9:1, v/v)
2+
+
+
Mn , Cs , Na , K
2+
Cu
2+
In 100% CH3CN, only Hg2+ induced large fluorescence enhancement
Pb
even though there were small fluorescent changes with Cu2+, Pb2+
,
520
540
560
580
600
620
640
and Zn2+ (Supplementary Fig. 8). From the fluorescent titration
experiments with Hg2+, the association constant was calculated
as 9.8 Â 104 MÀ1 (error <15%).13 Possibly, the binding of 2 with
Hg2+ can compete with that of water with Hg2+. Indeed, most
Wavelength (nm)
Figure 1. Fluorescent changes of 1 (3
lM) with various metal ions (150 lM) in
CH3CN–H2O (9:1, v/v) (kex = 510 nm, Slit: 3 nm/5 nm).

3288
S. K. Kim et al. / Tetrahedron Letters 51 (2010) 3286–3289
of Korea (NRF) and the Converging Research Center Program
through NRF funded by the Ministry of Education, Science and
Technology (2009-0093677). Mass spectral data were obtained
from the Korea Basic Science Institute (Daegu) on a Jeol JMS 700
high resolution mass spectrometer.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2010.04.067.
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10. Monoboronic acid-RB (1): Rhodamine ethylamine (4) (0.2 g, 0.4 mmol) was
dissolved in 20 mL of absolute ethanol. After 2-formylphenylboronic acid
(0.093 g, 0.62 mmol) was added, the reaction mixture was refluxed in an air
bath for 6 h. Then, the solution was cooled and concentrated to 10 mL and
allowed to stand at room temperature overnight. Ethanol was evaporated
completely under reduced pressure. The imine intermediate was dissolved in
10 mL of absolute methanol. Sodium borohydride was then added slowly with
stirring at room temperature for 4 h. The resulting solution was extracted with
methylene chloride (3 Â 100 mL), washed the organic layer with 50 mL of
Figure 6. Fluorescent changes (a) and colorimetric changes (b) of 2 (30 lM) with
Hg2+ (50 equiv) in CH3CN.
precedent rhodamine-based sensors bearing amide-linked binding
sites were studied in 100% organic solvent system or in the pres-
ence of small amount of aqueous system if they are reversible
probes.9 The UV absorption changes of 2 with Hg2+ in CH3CN are
also explained in the Supplementary data. On the other hand, in
the presence of buffer (pH 7.4) solution the selectivity for Hg2+
was enhanced dramatically (Supplementary Fig. 11). However,
the fluorescent enhancement process in this solvent system
[CH3CN–HEPES buffer (pH 7.4, 0.01 M) (9:1, v/v)] was also substan-
tially slowed down. The pH dependence of probe 2 was checked
(Supplementary Fig. 12) and the pKa of this probe was calculated
as around 2.4, which means that this probe has relatively a large
pH window between pH 6–9. Finally Figure 6 shows the fluores-
cent change and colorimetric change of 2 with Hg2+ in CH3CN.
The addition of Hg2+ induced a selective ‘Off–On’ fluorescent
change and distinct color change from colorless to dark pink.
In conclusion, rhodamine-boronic acid derivatives 1 and 2 were
synthesized as selective fluorescent and colorimetric sensors for
Hg2+ in aqueous solution. ‘Off–On’-type fluorescent and colorimet-
ric changes were observed for Hg2+, in which the spirolactam
(nonfluorescent) to ring-opened amide (fluorescent) process was
utilized. Most importantly, boronic acid group was utilized as a
binding unit for the Hg2+ selective fluorescent chemosensor for
the first time. The present study successfully demonstrated that
boronic acid group can serve as a unique and novel ligand for the
recognition of metal ions, which will be utilized for various recep-
tors in the future.
Acknowledgments
This work was supported by the Basic Science Research Pro-
gram (20090083065) through the National Research Foundation

S. K. Kim et al. / Tetrahedron Letters 51 (2010) 3286–3289
3289
water, dried on Na2SO4, and evaporated the solvents to get the crude product.
Purification by silica column chromatography (CH2Cl2–MeOH = 95:5, v/v)
afforded 0.10 g of 1 (41%) as pink solid. Mp: 152 °C; 1H NMR (CDCl3+MeOD
three drops, 250 MHz) d 7.78 (m, 1H), 7.42 (m, 2H), 7.27 (m, 1H), 7.12 (m, 2H),
7.05 (m, 1H), 6.95 (m, 1H), 6.37 (s, 1H), 6.33 (s, 1H), 6.30, (d, 2H, J = 2.5 Hz),
6.21 (dd, 2H, J = 8.9 Hz & 2.5 Hz), 3.33 (m, 12H), 2.73 (t, 2H, J = 6.4 Hz), 1.08 (t,
12H, J = 6.9 Hz); 13C NMR (CDCl3+MeOD three drops, 62.5 MHz) d 170.0, 153.5,
153.3, 149.0, 133.2, 130.2, 128.5, 128.3, 127.3, 126.7, 124.1, 122.8, 108.4, 104.1,
97.7, 66.1, 54.5, 44.3, 38.5, 12.6; HRMS (FAB) m/z = 619.3377 (M+H)+, calcd for
C37H44BN4O4 = 619.3373.
2H, J = 8.8 Hz & 2.5 Hz), 3.49 (m, 2H), 3.41 (s, 4H), 3.34 (s, 4H), 3.31 (q, 8H,
J = 7.0 Hz), 2.65 (t, 2H, J = 6.4 Hz), 1.10 (t, 12H, J = 7.0 Hz); 13C NMR (CDCl3) d
168.4, 153.4, 149.0, 138.4, 132.9, 130.8, 128.7, 128.5, 127.4, 124.1, 123.0, 108.2,
104.8, 97.8, 65.4, 61.4, 44.4, 34.9, 12.6; HRMS (FAB) m/z = 753.3916 (M+H)+,
calcd for C44H51BN4O6 = 753.3919.
12. Bisphenyl-RB (3): Application of procedure A to rhodamine ethylamine (0.13 g,
0.27 mmol) and benzyl bromide (0.092 g, 0.54 mmol) gave 0.075 g of 3 (54%) as
a light pink semi-solid after purification of the crude product on column (silica;
CH2Cl2–MeOH; 96:4). Mp: 168 °C; 1H NMR (CDCl3, 250 MHz) d 7.87 (m, 1H),
7.41 (m, 2H), 7.21 (m, 4H), 7.16 (m, 4H), 7.17 (m, 1H), 7.05 (m, 1H), 6.40 (s, 1H),
6.37 (s, 1H), 6.31 (d, 2H), 6.21 (dd, 2H, J = 8.8 Hz & 2.5 Hz), 3.44 (s, 4H), 3.35 (t,
2H), 3.30 (q, 8H), 2.31 (m, 2H), 1.17 (t, 12H, J = 7.0 Hz); 13C NMR (CDCl3,
62.5 MHz) d 167.8, 153.6, 153.2, 148.6, 139.3, 132.2, 131.4, 128.9, 128.0, 127.9,
126.6, 123.7, 122.6, 107.9, 105.6, 97.7, 64.8, 57.4, 51.4, 44.3, 38.1, 12.6; HRMS
(FAB) m/z = 665.3856 (M+H)+, calcd for C44H51BN4O6 = 665.3856.
11. Bis-boronic acid-RB (2): Procedure A. To a 250 mL flask, rhodamine ethylamine
(4) (0.3 g, 0.62 mmol) was dissolved in 30 mL of anhydrous chloroform. 0.86 g
(6.2 mmol) of anhydrous potassium carbonate was added followed with 0.4 g,
(1.86 mmol) 2-bromomethylphenylboronic acid. The reaction mixture was
stirred at room temperature until the disappearance of amine. Solvent was
evaporated and purified the crude product on column (silica; CH2Cl2–
MeOH = 96:4, v/v) to give 0.17 g of 2 (37%) as pink solid. Mp: 168 °C; 1H
NMR (CDCl3+MeOD three drops, 250 MHz) d 7.83 (m, 1H), 7.43 (m, 2H), 7.04
(m, 5H), 6.82 (m, 2H), 6.31 (s, 1H), 6.28 (s, 1H), 6.26, (d, 2H, J = 2.3 Hz), 6.20 (dd,
13. (a) Association constants were obtained using the computer program
ENZFITTER, available from Elsevier-BIOSOFT, 68 Hills Road, Cambridge CB2
1LA, United Kingdom.; (b) Conners, K. A.. Binding Constants. In The Measurement
of Molecular Complex Stability; Wiley: New York, 1987.