Rhodamine spirolactam sensors operated by sulfur-cooperated metal complexation

Article abstract of DOI:10.1016/j.saa.2017.07.019

New rhodamine Schiff base sensors were developed to improve selective sensing by introducing sulfide, ester, and dithiocarbonate groups, as well as using ketones coupled to rhodamine-hydrazine. Metal sensing proceeded through the 1:1 complexation of the metal ion for most sensors in the presence of Cu^2?+ and Hg^2?+. A sensor carrying a dithiocarbonate group responded selectively to Hg^2?+ showing a strong colorimetric change and intense fluorescence. The association constants of the sensors were determined from a linear plot performed at micro-molar concentrations to afford values in the range of 10⁴. Sensing was interrupted at the initial time of Hg^2?+ exposure due to the isomerization of imine and preferential metal bonding of two dithiocarbonate groups regardless of the main structure of rhodamine. The sensors exhibited the reversible and reproducible performance for Hg^2?+ sensing.

Full text of DOI:10.1016/j.saa.2017.07.019

Accepted Manuscript
Rhodamine spirolactam sensors operated by sulfur-cooperated
metal complexation
Gisuk Heo, Dahye Lee, Chi Gwan Kim, Jung Yun Do
PII:
S1386-1425(17)30578-4
doi: 10.1016/j.saa.2017.07.019
SAA 15306
DOI:
Reference:
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date:
Revised date:
Accepted date:
28 February 2017
14 July 2017
14 July 2017
Please cite this article as: Gisuk Heo, Dahye Lee, Chi Gwan Kim, Jung Yun Do ,
Rhodamine spirolactam sensors operated by sulfur-cooperated metal complexation,
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2017), doi:
1
0.1016/j.saa.2017.07.019
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ACCEPTED MANUSCRIPT
Rhodamine Spirolactam Sensors Operated by Sulfur-cooperated Metal Complexation
1
2
1
1,2,
Gisuk Heo , Dahye Lee , Chi Gwan Kim , Jung Yun Do *
1
Department of Chemical Materials, Pusan National University, Busandaehak-ro 63beon-gil,
Geumjeong-gu, Busan, 46241, Republic of Korea
2
Department of Chemistry Education, Pusan National University, Busandaehak-ro 63beon-
gil, Geumjeong-gu, Busan, 46241, Republic of Korea
*
Corresponding author. fax: +82 51 581 2348; E-mail: jydo@pusan.ac.kr
1

ACCEPTED MANUSCRIPT
Abstract: New rhodamine Schiff base sensors were developed to improve selective sensing
by introducing sulfide, ester, and dithiocarbonate groups, as well as using ketones coupled to
rhodamine-hydrazine. Metal sensing proceeded through the 1:1 complexation of the metal ion
2
+
2+
for most sensors in the presence of Cu and Hg . A sensor carrying a dithiocarbonate group
2
+
responded selectively to Hg showing a strong colorimetric change and intense fluorescence.
The association constants of the sensors were determined from a linear plot performed at
4
micro-molar concentrations to afford values in the range of 10 . Sensing was interrupted at
2
+
the initial time of Hg exposure due to the isomerization of imine and preferential metal
bonding of two dithiocarbonate groups regardless of the main structure of rhodamine. The
2
+
sensors exhibited the reversible and reproducible performance for Hg sensing.
Keywords:
2
+
Rhodamine, Hg sensing, association constant, dithiocarbonate, reversible sensing
2

ACCEPTED MANUSCRIPT
1
. Introduction
The selective and sensitive detection of metal ions has been a major goal in research
involving very low concentrations, such as the natural environment, industrial waste, human
body, food, etc [1-3]. Several sensors have been developed through artificial molecular
technology for sensing transition and heavy metal ions [3-7]. The easy synthesis of sensor
molecules, sensing tolerance, and detection limit are current research issues. The metal
recognition of sensing molecules involves the kinetic and thermodynamic motions of the
molecules for stable complex formation or loosened binding. The spiro-ring opening of
rhodamine lactam based molecules is a constant mechanism of established metal sensors
[
8,9]. The metal-promoted color development of a colorless spiro rhodamine facilitated
naked-eye detection and provided an extremely low detection limit. The convenient
formation of a Schiff base was used to develop several rhodamine sensors, in which the
preferential binding sites of N, O, imide N, and spiro-carbonyl bound cooperatively to metals,
2
+
3+
2+
including Cu , Al , and Hg [2]. Hong, who designed a rhodamine derivative carrying S,S`-
2
+
diallyl carbohydrazonodithioate, reported the highly selective and rapid response of a Hg
sensor [10]. The sensor exhibited strong fluorescence through an irreversible reaction
2
+
2+
operated by Hg . The Hg sensor was developed based on the strong thiophilic nature of the
mercury ion [10-13]. The rhodamine analog derived from simple O/S replacement exhibited
2
+
excellent Hg sensing properties, which resulted from stable complex formation with a 1:2
2
+
binding ratio (Hg /rhodamine) [14]. Yoon et al. reported [15] similar 1:2 complex formation
to allow the highly selective Hg sensing of rhodamine hydrazone bearing a thiol group. For
the better Hg sensing, the present study focus on extending the binding sites in a rhodamine
molecule and allow the formation of a 1:1 complex with multiple S binding sites, which
2
+
would enhance Hg recognition. Several thiocarbonyl groups are examined to involve in the
3

ACCEPTED MANUSCRIPT
formation of the Hg-rhodamine complex. Metal selectivity from binding contribution of
dithiocarbonyls is synthetically controllable with simple electronic modification. The
cooperative binding of S or O along with dithiocarbonate leads to the formation of a 1/1
metal complexation and enhances metal association of previous unselective rhodamine
sensors [9]. The sensing property altered by peculiar binding groups was investigated through
the spectroscopic determination of the association constants.
2
. Experimental detail
2
.1 Material and instrumentations
The starting materials and reagents were used as purchased from Sigma-Aldrich
Corporation, and the reagent-grade solvents were dried when necessary and purified by
vacuum distillation. Column chromatography for the purification of products was performed
1
13
using silica gel (Merck, 250-430 mesh). H NMR and C NMR spectra were recorded in
CDCl for internal reference on a Varian Mercury 300 MHz and 75 MHz, respectively. The
3
mass spectra were recorded on an Agilent 1200LC/1100 MSD SL mass spectrometer. The
UV-Vis spectra were measured using a Jasco V-570 UV-Vis spectrometer. The absorption
spectra were recorded from 250 to 800 nm using a path length of 10 mm, a bandwidth of 1.0
nm, and a scan speed of 250 nm/min. Fluorescence spectra were recorded in a Hitach F-4600
fluorescence spectrometer at room temperature and the excitation and emission slit widths
both were 5.0 nm.
2
+
2+
2+
2+
2+
2+
3+
Stock solutions (0.01 M) of Ca , Co , Cu , Zn , Cd , Hg , and Cr were prepared
using CaCl , Co(NO ) 6H O, Cu(ClO ) , ZnCl , CdCl , Pb(NO ) , HgCl and Cr(NO ) 9H O,
2
3 2
2
4 2
2
2
3 2
2
3 3
2
-4
respectively, in distillated water and diluted with ethanol (1/9, v/v) to give the range of 10 -
-6
1
0
M concentration. All diluted solutions were buffered using 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES, 99.5%) of 1.0 x 10 mM concentration at pH 7.4.
4

ACCEPTED MANUSCRIPT
2
2
.2 Synthesis
.2.1 Synthesis of ketone 1 (general procedure)
-Buthanethiol (2.1 mL, 20 mmol, 99%) and chloroacetone (2.0 mL, 24 mmol, 95%) were
1
dissolved in dimethlyformamide (30 mL). After addition of potassium carbonate (3.0 g, 22
mmol), the mixture was stirred at room temperature for 8 h. The resulting mixture was
diluted with water and extracted with ether twice. The ether layer was dried over anhydrous
MgSO and concentrated by rotary evaporator. A column chromatography on silica gel was
4
1
performed using ethyl acetate / hexane (1/7, v/v) to obtain ketone 1 of 2.49 g (85 %). H
NMR (300 MHz, CDCl ) δ 3.22 (s, 2H), 2.48 (t, J=7.2 Hz, 2H), 2.79 (s, 3H), 1.54 (m, 2H),
3
1
3
1
2
.40 (m, 2H), 0.91 (t, J=7.2 Hz, 3H). C NMR (75 MHz, CDCl ) δ 202.3, 41.0, 30.9, 30.3,
3
6.7, 21.1, 12.9.
1
Ketone 2 (57 % isolation yield): H NMR (300 MHz, CDCl ) δ 3.46–3.36 (m, 3H), 3.16 (d,
3
1
3
J=7.0 Hz, 2H), 2.96 (d, J=7.3 Hz, 2H), 1.97 (d, J=8.0 Hz, 3H). C NMR (75 MHz, CDCl ) δ
3
2
02.4, 169.4, 51.5, 41.4, 32.3, 27.4. (see supplementary date for synthesis in detail)
1
Ketone 3 (55 % isolation yield): H NMR (300 MHz, CDCl ) δ 8.93 (s, 1H), 3.39 (s, 2H),
3
1
3
3
.19 (s, 2H), 2.21 (s, 3H). C NMR (75 MHz, CDCl ) δ 204.2, 174.0, 41.8, 32.9, 28.1.
3
1
Ketone 4 (92 % isolation yield): H NMR (300 MHz, CDCl ) δ 4.64 (q, J=7.1 Hz, 2H), 4.00
3
1
3
(
s, 2H), 2.33 (s, 3H), 1.42 (t, J=7.1 Hz, 3H). C NMR (75 MHz, CDCl ) δ 213.0, 200.9, 70.7,
3
4
6.0, 29.1, 13.7.
1
Ketone 5 (34 % isolation yield): H NMR (300 MHz, CDCl ) δ 4.64 (s, 2H), 4.22 (q, J=7.1
3
1
3
Hz, 2H), 2.16 (s, 3H), 1.32 (t, J=7.1 Hz, 3H). C NMR (75 MHz, CDCl ) δ 201.4, 154.6,
3
7
0.6, 64.6, 14.3, 13.9.
5

ACCEPTED MANUSCRIPT
1
Ketone 6 (93 % isolation yield): H NMR (300 MHz, CDCl ) δ 4.16 (s, 2H), 4.07–3.65 (m,
3
1
3
4
2
H), 2.30 (s, 3H), 1.31–1.19 (m, 6H). C NMR (75 MHz, CDCl ) δ 202.2, 193.7, 49.9, 46.6,
3
9.2, 12.2.
1
Ketone 7 (61 % isolation yield): H NMR (300 MHz, CDCl ) δ 4.61 (q, J=7.1 Hz, 4H), 4.17
3
1
3
(
s, 4H), 1.40 (t, J=7.1 Hz, 4H). C NMR (75 MHz, CDCl ) δ 212.6, 196.2, 70.9, 44.7, 13.0.
3
2
.2.2 Synthesis of rhodamine-hydrazine
To a stirred solution of rhodamine B (4.8 g, 10.0 mmol, 97%) in ethanol (30 mL) was added
o
hydrazine hydrate (3.7 mL, 117.0 mmol, 99%). The mixture was refluxed at 85 C for 4 h,
cooled to room temperature, and solvent was removed by rotary evaporator under reduced
pressure. The mixture was purified by column chromatography on silica gel using ethyl
1
acetate / hexane (1/1, v/v) to yield white solid 3.3 g (73 %). H NMR (300 MHz, CDCl ) δ
3
7
4
1
1
.99–7.89 (m, 1H), 7.51–7.38 (m, 2H), 7.10 (dd, J=5.6, 2.5 Hz, 1H), 6.44 (dd, J=11.7, 5.6 Hz,
H), 6.29 (dd, J=8.8, 2.5 Hz, 2H), 3.61 (s, 2H), 3.34 (q, J=7.0 Hz, 8H), 1.16 (t, J=7.0 Hz,
1
3
2H). C NMR (75 MHz, CDCl ) δ 166.0, 153.7, 151.4, 148.7, 132.4, 129.9, 128.0, 123.7,
3
22.8, 107.8, 104.3, 97.8, 65.8, 44.2, 12.5.
2
.2.3 Synthesis of rhodamine derivative D1 (general procedure)
In a two-necked round bottomed flask of 25 mL equipped with a reflux condenser,
rhodamine-hydrazine (2.74 g, 6.0 mmol) and the prepared ketone 1 (0.97 g, 6.6 mmol) were
placed and charged with benzene (18 mL). The mixture was refluxed for 24 h. The solvent
was removed by rotary evaporator under reduced pressure and resulting mixture was purified
by column chromatography on silica gel using ethyl acetate / hexane (1/2, v/v) to afford
1
powder of 2.63 g (75%). H NMR (300 MHz, CDCl ) δ 7.90 (s, 1H), 7.40 (m, 2H), 7.08 (m,
3
6

ACCEPTED MANUSCRIPT
1
2
H), 6.53 (d, J=8.7 Hz, 2H), 6.34 (s, 2H), 6.24 (d, J=8.7 Hz, 2H), 3.29 (m, 8H), 3.13 (s, 2H),
1
3
.0 (m, 5H), 1.32-1.20 (m, 4H), 1.13 (m, 12H), 0.85 (t, J=7.2 Hz, 3H). C NMR (75 MHz,
CDCl ) δ 171.6, 160.2, 153.6, 148.1, 132.0, 130.2, 128.2, 127.8, 123.5, 122.5, 107.4, 105.6,
3
9
7.3, 65.9, 44.0, 37.4, 30.4, 28.7, 21.2, 17.9, 13.6, 12.2. Liquid chromatography–mass
+
spectrometry (LC–MS): calculated exact mass: 584.32, found 585.35 [M+H ].
1
D2 (65% isolation yield): H NMR (300 MHz, CDCl ) δ 7.91 (s, 1H), 7.54–7.41 (m, 2H),
3
7
3
.15 (s, 1H), 6.52 (d, J=8.8 Hz, 2H), 6.35 (s, 2H), 6.28 (d, J=8.9 Hz, 2H), 3.64 (s, 3H), 3.43–
1
3
.18 (m, 10H), 2.86 (s, 2H), 1.96 (s, 3H), 1.16 (t, J=6.8 Hz, 12H). C NMR (75 MHz,
CDCl ) δ 170. 5, 170.2, 159.9, 153.1, 151.2, 148.1, 132.0, 130.2, 128.0, 127.8, 123.5, 122.5,
3
1
6
07.4, 105.5, 97.4, 66.0, 51.7, 43.9, 38.2, 30.6, 18.2, 12.2. LC–MS: calculated exact mass:
+
00.28, found 601.30 [M+H ].
1
D3 (56% isolation yield): H NMR (300 MHz, CDCl ) δ 8.07 (s, 1H), 7.60 (s, 2H), 7.14 (s,
3
2
H), 6.65 (s, 4H), 6.48 (s, 2H), 3.60 (s, 2H), 3.46 (q, J=5.4 Hz, 8H), 3.11 (s, 2H), 2.45–2.03
+
(
m, 3H), 1.27 (s, 12H). LC–MS: calculated exact mass: 586.26, found 585.26 [M-H ].
1
D4 (60% isolation yield): H NMR (300 MHz, CDCl ) δ 7.91 (s, 1H), 7.46 (d, J=3.0 Hz, 2H),
3
7
2
.15 (s, 1H), 6.51 (d, J=7.8 Hz, 2H), 6.39 (s, 2H), 6.28 (d, J=8.5 Hz, 2H), 4.56 (q, J=6.3 Hz,
H), 3.97 (s, 2H), 3.34 (q, J=6.5 Hz, 8H), 1.92 (s, 3H), 1.35 (t, J=5.4 Hz, 3H), 1.16 (t, J=6.2
13
Hz, 12H). C NMR (75 MHz, CDCl ) δ 213.8, 169.8, 160.7, 153.4, 151.4, 148.4, 132.4,
3
1
30.0, 128.2, 128.1, 123.8, 122.8, 107.7, 105.9, 97.6, 70.1, 66.5, 45.8, 44.1, 22.0, 13.5, 12.5.
+
LC–MS: calculated exact mass: 616.25, found 617.28 [M+H ].
1
D5 (43% isolation yield): H NMR (300 MHz,CDCl ) δ 7.92–7.89 (m, 1H), 7.45 (dd, J=5.4,
3
3
4
1
.1 Hz, 2H), 7.14 (d, J=2.8 Hz, 1H), 6.52 (d, J=8.8 Hz, 2H), 6.38 (s, 2H), 6.28 (d, J=2.1, 2H),
.67 (s, 2H), 4.14 (q, J=7.0 Hz, 2H), 3.33 (q, J=6.9 Hz, 8H), 1.92 (s, 3H), 1.25 (t, J=6.9, 3H),
1
3
.16 (t, J= 6.8Hz, 12H). C NMR (75 MHz, CDCl ) δ 169.0, 160.3, 154.3, 153.4, 151.8,
3
7

ACCEPTED MANUSCRIPT
1
1
48.5, 132.3, 130.1, 128.4, 128.1, 107.4, 106.0, 97.9, 97.5, 69.7, 66.4, 64.2, 44.2, 13.9, 12.7,
+
2.4. LC–MS: calculated exact mass: 584.30, found 585.33 [M+H ].
1
D6 (77% isolation yield, isomers): H NMR (300 MHz, CDCl ) δ 7.90 (dd, J=5.6, 3.0 Hz,
3
1
H), 7.46 (dd, J=5.4, 3.0 Hz, 2H), 7.19–7.08 (m, 1H), 6.52 (d, J=6.2 Hz, 2H), 6.39 (d, J=2.1
Hz, 2H), 6.34–6.23 (m, 2H), 4.30 (s, 2H), 4.08–3.63 (m, 4H), 3.34 (q, J=6.7 Hz, 8H), 1.92 (s,
1
3
3
1
1
H), 1.23 (t, J=7.2 Hz, 3H), 1.17 (t, J=6.9 Hz, 12H). C NMR (75 MHz, CDCl ) δ 194.6,
3
71.8, 165.7, 159.9, 153.6, 153.3, 151.3, 148.6, 148.3, 130.4, 127.9, 122.8, 107.6, 106.0,
04.2, 97.7, 66.2, 65.7, 49.9, 46.6, 44.1, 12.4, 11.4. LC–MS: calculated exact mass: 643.30,
+
found 644.34 [M+H ].
1
D7 (65% isolation yield, isomers): H NMR (300 MHz, CDCl ) δ 7.92 (d, J=6.1 Hz, 1H),
3
7
2
1
1
1
.52 (d, J=3.3 Hz, 2H), 7.22 (s, 1H), 6.47 (d, J=8.7 Hz, 2H), 6.41 (s, 2H), 6.28 (d, J=8.7 Hz,
H), 4.59–4.46 (m, 4H), 4.06 (d, J=24.9 Hz, 4H), 3.35 (q, J=6.8 Hz, 8H), 1.51–1.22 (m, 6H),
1
3
.19 (t, J=6.6 Hz, 12H). C NMR (75 MHz, CDCl ) δ 213.1, 164.4, 160.7, 153.6, 151.0,
3
48.5, 132.7, 130.2, 128.3, 128.2, 124.1, 123.1, 107.7, 106.1, 97.7, 70.2, 67.2, 44.2, 36.5,
+
3.5, 12.5. LC–MS: calculated exact mass: 736.22, found 737.25 [M+H ].
2
.3 General sample preparation for the measurement of absorption and fluorescence spectra
-3
Sensor 4 was dissolved in EtOH to make a 1.0 x 10 M stock solution. Cu(ClO ) 6H O
4
2
2
-3
2+
(
98%) was dissolved in distilled water to make a 1.0 x 10 M stock solution of Cu . To a 10
2
+
mL glass tube containing 1.0 mL of HEPES (0.10 M in EtOH), Cu solution (1.0 mL) and
sensor solution (0.1 mL) were added, and diluted with EtOH (8.9 mL) to make water-EtOH
solvent in a 1/9 ratio (v/v). After keeping for 10 min, 1.0 mL of the mixed solution was
transferred to another 10 mL glass tube containing 9.0 mL of water-EtOH (1/9) and HEPES
(1.0 x 10 mM) to make a 1.0 x 10 M-sensor concentration for optical analysis. The sensor
8

ACCEPTED MANUSCRIPT
exposure upon metal ion was performed for 10 min before final dilution for the purpose of
achieving a fast equilibrium. That was equally applied to all sensing samples unless otherwise
specified.
The experiment of dynamic sensing property used samples which were prepared through
immediate dilution to make a final concentration without a delay before measurement.
3
. Results and Discussion
3
.1. Synthesis
Rhodamine derivatives were synthesized through the formation of a Schiff base from the
condensation of rhodamine-hydrazine with a ketone [2,16]. Most ketones in the study were
derived starting from chloroacetone through the simple alkylation of thiols or metal thiolates.
The alkylation of n-buthanethiol mediated by K CO afforded ketone 1 in N,N-
2
3
dimethylformamide. The reaction of methyl thioglycolate and chloroacetone generated
ketone 2 and the successive basic hydrolysis yielded ketone 3. Carbonodithiolate 4 and
carbamodithioate 5 were prepared by alkylation using potassium ethyl xanthate and sodium
diethyl dithocarbamate, respectively, in more than 95% yield. The esterification of
hydroxyacetone with chloro ethylfomate produced ketone 5 as an oxygen analog of ketone 4.
The double alkylation of 1,3-dichloroacetone using potassium ethyl xanthate in ethanol
afforded symmetric ketone 7, as shown in figure 1.
When the prepared ketone was heated under reflux in benzene with rhodamine-hydrazine
[
2], a new rhodamine Schiff base was generated through a coupling reaction and is shown in
figure 2 as rhodamine sensors D1 – D7 depending on starting ketone. The resulting sensors
were isolated and used in an isomeric mixture because of the rapid interconversion of every
isolated molecule reproducing the original composition over 10 min. Isomerization may
9

ACCEPTED MANUSCRIPT
2
occur through free rotation along the N-N bond and cis-trans flip on the sp N atom [17,18].
This reversible isomerization phenomenon might shift the equilibrium of metal complexation
through the conformational rearrangement of binding sites, such as alkyl groups attached to
imine, allowing multiple binding of a sensor molecule and consequently contribute to sensing
enhancement. Despite the isomerization contribution, sensor D7 was designed based on a
consideration that the alkyl group of symmetric ketone 7 can essentially achieve an
orientation suitable for the corresponding metal complexation, which is irrespective of the
1
13
isomerization of D7. The structure of the prepared sensors were analyzed by H and C
nuclear magnetic resonance (NMR) spectroscopy and confirmed by mass spectrometry.
3
.2. Optical analysis of metal sensing
Previous success in metal sensing has been suggested by complex formation of 1:1 or 1:2
binding stoichiometry of the metal to sensor [10-15]. The ratio was generally determined by a
Job`s plot, which plots the physical properties as a function of the mole fraction of the metal
or sensor [19]. A preliminary examination of the prepared rhodamine sensor exhibited strong
2
+
Cu binding by the opening spiro ring in D1 via metal ion binding at sites, such as N, O, or S
2
+
atoms. The colorless spirolactam D1 exhibited a bright color in the presence Cu and
2
+
2+
2+
unchanged in the presence of metals, such as Pb , Hg , and Cd . The UV-visible absorption
2
+
spectrum of D1 exposed to Cu (Cu-D1) exhibited a maximum absorption at approximately
2+
5
60 nm. The maximum intensity was recorded as an increased mole fraction of Cu while
2
+
the total molar concentration of Cu and D1 was kept at 5 M in H O/EtOH (1:9, v/v)
2
containing HEPES (10 mM). The H O/EtOH mixture was chosen as a solvent to enhance the
2
solubility of both the metal and sensor, and HEPES was introduced to maintain the pH
balance of the solution because rhodamine sensors are sensitive to pH. The absorption height
1
0

ACCEPTED MANUSCRIPT
2
+
was optimized to a ratio of 0.3, as shown in figure 3 (a). The Cu fraction of 0.3 indicates a
2
+
2+
mole ratio of Cu to the sum of Cu and D1. Therefore, complex Cu-D1 has a 1:2 binding
stoichiometry. The previous rhodamine hydrazine sensor was proposed as a 1:2 complex via
metal binding at the N and O atoms [15]. Therefore, the S atom of D1 is assumed to be
uncooperative in the formation of the complex Cu-D1. The other sensors (D2, D3, D4, D5,
D6, and D7) exhibited typical behavior of the complex formation with a 1:1 binding
stoichiometry and an optimal peak at a 0.5 mole fraction (see supplementary data). For
2
+
2+
example, a Job`s plot of D4 represented the 1:1 complex formation by Cu and Hg , as
2
+
shown in figure 3(b). The absorption intensity of Cu solutions was recorded at 560 (565 nm
2
+
for Hg solution). Total concentration of D4 and metal ion was 5.0 x 10 M and kept
constantly, while the mole fraction of metal ion changed from 0.2 to 0.9. Maximum
absorption was observed when the fraction was ca. 0.5, indicating 1:1 stoichiometry of metal
ion and D4. Sensor D4 carries an additional binding site of a dithiocarbonate group, in which
one of the S atoms is involved in 1:1 complexation. The third anion or solvent species might
satisfy the typical four coordination number of these metals in solution. The response of D4
2
+
to a soft metal, such as Hg , indicates the cooperative participation of the dithiocarbonate
group. The intense absorption of D4 at 565 nm and 560 nm was observed upon the addition
2
+
2+
of Hg and Cu , respectively, which induced a color change from colorless to pink. None of
3
+
2+
2+
2+
2+
the other metal ions (Cr , Ca , Co , Cd , and Zn ) were detectable by the naked eye and
in the corresponding absorption spectrum, as indicated in figure 4(a). The measurement was
performed with a sensor of 10 M in H O/EtOH (1:9, v/v) in the presence of 10 equiv. of
2
2+
metal ions. D7, which carries two dithiocarbonyl groups exhibited strong binding to Cu and
2
+
Hg . The similar metal selectivity of D4 and D7 was attributed to the contribution from the
dithiocarbonyl group. Furthermore, the enhanced absorption intensity of D7 compared to D4
1
1

ACCEPTED MANUSCRIPT
can be explained by the suitable orientation of a dithiocarbonyl group ready for binding metal
ions, regardless of the conformational rearrangement of the Schiff base. Despite carrying a
2
+
dithiocarbonyl group, sensor D6 is interesting because of its negligible Hg response. Metal
complexation of sensor D6 appears to be improved by a C=S binding site for which is
electron-enriched more by Et N than OEt of a xanthate group. Sensor D6 selectively
2
2
+
2+
responded upon Cu and was insensitive to Hg . Optical sensing of all the prepared sensors
D1-D7) was conducted toward various types of metal ions and compared using molar
absorption coefficients, as summarized in table 1. All sensors showed a strong response to
(
2
+
3+
2+
2+
2+
2+
Cu with an insignificant response to other metals, such as Cr , Ca , Co , Cd , and Zn .
2
+
Hg was responded strongly by D4 and D7 but weakly by other sensors. Rhodamine sensor
D5 is an oxygen analog of sensor D4, changing from S to O in the structure. Low absorbance
2
+
of D5 upon Hg verifies the important role of the S binding site of dithiocarbonate as a
critical parameter that influences Hg-binding.
3
.3 Fluorescence sensing of metal ions
The fluorescence spectra were recorded by excitation of the sensors (5.0 x 10 M in
H O/EtOH, 1:9) at 510 nm. Among all the sensors examined, D4 exhibited the highest
2
2
+
2
intensity of emission in the presence of Hg (5.0 x 10 M), as shown in figure 4(b). The
weak fluorescence of D7 was observed, despite the fact that the absorption of D7 was higher
2
+
than that of D4 upon exposure to Hg . An unknown quenching mechanism, possibly energy
transfer toward a free-dithiocarbonyl group in Hg-D7, is believed to be involved [20]. The
observed fluorescence intensity of Hg-D4 and Hg-D7 was constant over 24 h after mixing.
Despite the strong absorption of Cu-D4 and Cu-D7, the fluorescence was barely observed,
2
+
suggesting the strong contribution of the paramagnetic Cu quenching effect [16].
1
2

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3
.4 Association constants
All the sensors except for D1 underwent metal complexation at 1:1 stoichiometry, as
determined by the Job’s plot. The association constant was calculated using the Benesi-
Hildebrand method from the titration profile based on 1:1 binding [19,21].
Where A and A are the absorbance of the sensor solution in the presence and absence of
0
metal ions, respectively; Amax is the saturated absorbance obtained with an excess of metal
-1
ions. The association constant (K) was determined from a linear plot of (A-A ) , as a
0
n+ -1
function of the reciprocal molar concentration, [M ] , of the metal ion. The association
constants of the six sensors were determined as shown in table 2 (see supplementary data for
determining a constant). All sensors exhibited a constant in the range of four orders of
magnitude, representing strong sensing property. The association constants of sensor D7 were
2
+
2+
twice larger than those of D4 upon both Hg and Cu . This result was attributed to a
favorable arrangement for metal binding caused by two dithiocarbonate branches.
3
.5 Dynamic sensing property of complex formation
Most metal binding occurred rapidly and equilibrium was established in a few minutes,
affording the maximum absorption intensity. On the other hand, the complexation of D4 and
2
+
D7 to Hg occurred gradually in the initial period and was complete after an hour, which was
observed by the change of absorbance at a 10 M concentration. The curve of D4 in figure 5
is composed of several nonlinear growths, before reaching saturation, in which metal binding
can be interrupted by isomerization along the C=N bond of the dithiocarbonate group (figure
1
3

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2
+
2+
6
(a)). Sensor D7 exhibited fast and slow saturation curves upon Cu and Hg , respectively
figure 5), involving the specific behavior at the initial period (“A” circle in figure 5). The
kinetic behavior of the complexation of D7-Hg appears to proceed via two steps, as proposed
(
2
+
in figure 6(b). Two dithiocarbonate groups seize Hg in the pre-induction step and
subsequent ring-opening occur. The initial delay curve was not observed in D4 and even
disappeared at above a 20 M concentration of D7, causing rapid saturation.
The reversible sensing performance was evaluated by EDTA (ethylenediaminetetraacetic
2
+
acid) titration. The strong affinity of EDTA to Hg can liberate D4 from the D4-Hg complex.
The absorbance intensity decreased upon addition of EDTA to a solution of D4-Hg complex.
The liberation of D4 occurred incompletely to retain the minimum absorbance, even in excess
2
+
EDTA. By adding Hg again, the absorbance recovered, indicating the reversible metal
2
+
binding of D4. The simple addition of Hg to a mixture composed of D4 and EDTA resulted
+
in partial formation of the D4-Hg complex. In figure 7, a mixture of EDTA/Hg at a 1:1 mole
ratio presents the different absorbance of 0.29 and 0.12 for titration and simple addition,
respectively. Other comparative experimental observations showed similar results.
Consequently, the liberation of D4 from D4-Hg occurred slowly and the formation of D4-Hg
proceeded rapidly, meaning the fast sensing of D4.
4
. Conclusions
Rhodamine Schiff base sensors carrying dithiocarbonate group were first developed and
2
+
evaluated by selective Hg sensing. Cooperative metal complexation occurred depending on
the adjacent binding sites, such as sulfide, carboxylate, carbonate, and dithiocarbonate as well
2
+
as the nitrogen atom of lactam and imine. Most of the prepared sensors responded to Cu
2
+
and the presence of dithiocarbonate group contributed to the strong Hg binding. Based on
1
4

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1
:1 complexation of the sensor vs. metal ion, the association constant was determined from
the linear response to metal ions in micro-molar concentrations; the values were in the range
of four orders of magnitude. The rhodamine sensor carrying dithicarbonate exhibited intense
2
+
fluorescence through selective Hg binding, which was reversible and reproducible, and
2
+
allowed visual recognition, making it a practical Hg sensor. New sensors based on metal
chelation will be improved by introducing binding sites possessing a metal preference,
leading to selective sensing.
Acknowledgement: This subject was supported by Korea Ministry of Environment (MOE) as
“
Chemical Accident Prevention Technology Development Project”
1
5

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Legends of tables and figures
Table 1. Molar absorption coefficient of all the prepared sensors toward metal ions
Table 2. Association constant of the sensors
Figure 1. The synthesis of ketones (1-7)
Figure 2. The synthesis of rhodamine Schiff base sensors (D1-D7)
Figure 3. The Job`s plot curves of the sensor D1(a) and D4(b) for determining stoichiometry
2
+
of a metal complex through measuring maximum absorption intensity of Cu and
2
+
Hg at 560 nm and 565 nm, respectively.
Figure 4. (a) The UV-vis absorption spectrum of the sensor D4 and D7 upon various metal
ions at a 10 M H O/EtOH (1:9, v/v) and (b) fluorescence spectrum of sensors at a
2
2
+
2+
5
0 M H O/EtOH (1:9, v/v) upon Cu or Hg (500 M) excited at 510 nm.
2
Figure 5. The absorption change of the sensor D4 and D7 in 10 M H O/EtOH (1:9, v/v)
2
containing 10 equiv. of the metal ions with a function of sensing time.
2
+
Figure 6. (a) Conformational equilibrium of D4 and (b) preferential Hg binding seized by
two dithiocarbonate groups before ring opening of a rhodamine sensor D7.
2
+
Figure 7. The reversible sensing of D4 to Hg performed by adding Na EDTA and
2
2
+
successive adding Hg .
1
9

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Figure 1
O
O
O
O
RSH
base
ClCO2Et
OH
Cl
OCO2Et
S
Et N
R
3
5
R = n-Bu
1
2
3
4
6
O
O
CH CO Me
2
2
CH CO H
KSC(S)OEt
2
2
S
S
S
S
CH2SC(S)OEt
CH2SC(S)NEt2
Cl
Cl
OEt
OEt
7
2
0

ACCEPTED MANUSCRIPT
Figure 2
Et N
O
NEt2
Et N
2
O
NEt2
2
benzene
reflux, 24h
+
ketones
N NH2
O
N N
O
CH R
2
CH R`
2
Rhodamine-hydrazine
(
R=H) R`=
SC H
(R=H)R`=
R=R`=
SC(S)OEt
SC(S)OEt
D1
D2
D3
4
9
D4
D5
D7
SCH CO Me
OCO Et
2
2
2
SCH CO H
D6 SC(S)NEt2
2
2
2
1

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Figure 3
2
2

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Figure 4
2
3

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Figure 5
0.8
0.6
0.4
0.2
0.0
Hg(II)-D7, 565 nm
Cu(II)-D7, 560 nm
Hg(II)-D4, 565 nm
A
0
0.05 0.1 0.3 0.5 0.7 0.8
time (hour)
1
4
12 24 48
2
4

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Figure 6
2
5

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Figure 7
2
6

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Table 1 Molar absorption coefficient of all the prepared sensors toward metal ions
4
1
Absorbance ( ε x 10 )
2
D1
D2
9.1
0.02
u
D3
7.5
0.03
u
D4
3.4
4.30
u
D5
2.3
0.06
u
D6
3.9
0.02
u
D7
5.3
6.40
u
sensor
2
+
1
2.1
Cu
Hg
2
+
0
.02
n+3
4
M
u
1Determined from maximum absorption intensity at 560 nm and 565 nm for Cu2+ and Hg2+,
2
respectively. 10 Equiv. of metal ions was introduced to a sensor in 10 mM H O/EtOH (1:9, v/v)
2
3
n+
2+
2+
2+
2+
3+ 4
containing PETES (10 mM). M includes Zn , Ca , Pb , Cd and Co . undetermined (less
than detection limit).
2
7

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Table 2. Association constant of the sensors
4
1
Association constant ( K x 10 )
sensor
D2
D3
2.7
u
D4
1.7
1.3
D5
1.8
u
D6
5.8
u
D7
3.4
3.2
2+
2
.2
Cu
Hg
2+
2
u
1Determined from linear plot of maximum absorbance against metal concentration, measured at
60 nm and 565 nm for Cu2+ and Hg , respectively. Undeterimned due to low Hg response of
2+
2
2+
5
D2,D3,D5 and D6.
2
8

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Rhodamine Spirolactam Sensors Operated by Sulfur-cooperated Metal Complexation
*
Gisuk Heo, Dahye Lee, Chi Gwan Kim, Jung Yun Do
Graphical abstract
2
9