A BODIPY-based reactive probe for ratiometric fluorescence sensing of mercury ions

Article abstract of DOI:10.1016/j.tet.2012.01.091

A simple BODIPY derivative is demonstrated to fluorescently sense Hg ²⁺ in a ratiometric manner. The probe, an 8-methylthio-BODIPY, undergoes Hg²⁺-promoted hydrolysis to produce the corresponding 8-hydroxy-BODIPY, which conversion is accompanied with a large emission wavelength change. The probe can selectively detect Hg²⁺ over various other metal cations, with a detection limit of 1 ppb.

Full text of DOI:10.1016/j.tet.2012.01.091

Supplementary Data
For
A BODIPY-based reactive probe for ratiometric
fluorescence sensing of mercury Ions
Dokyoung Kim,a Koji Yamamoto,b Kyo Han Ahna,*
aDepartment of Chemistry and the Center for Electro-Photo Behaviors in Advanced Molecular Systems,
b
POSTECH, San 31, Hyoja-dong, Pohang, 790-784, Republic of Korea. Department of Pharmaceutical
Sciences, Tohoku University, Aoba-ku, Sendai 980-8578, Japan.
*To whom correspondence should be made:
Professor Kyo Han Ahn
Tel: +82) 54-279-2105
Fax: +82) 54-279-3399
Email: ahn@postech.ac.kr
CONTENT
General Methods and Synthesis of probe 1.
Figure S1. Time-dependent absorbance and fluorescence emission spectra profile of the probe 1 (10 μM)
upon addition of HgCl2 (1:1 equiv.) in the 10% CH3CN/HEPES buffer (10 mM, pH 7.4).
Figure S2. Absorbance and fluorescence emission data obtained for a 1:1 mixture of probe 1 (10 μM)
and each of the various metal ions (Hg2+, Ni2+, Ba2+, Cr2+, Au3+, Pb2+, Mn2+, Ag+, Co2+, Al3+,
Cd2+, Fe3+, Cu2+, Pd2+, Mg2+, Ca+, and Zn2+; as the chloride salts) in the 10% CH3CN/HEPES
buffer (10 mM, pH 7.4), acquired after 180 min.
Figure S3. A plot of fluorescence intensity depending on the concentration of HgCl2 in the range of
0.25–100 ppb.
Figure S4. (a) Fluorescence spectra of probe 1 (10 μM) in the presence of different concentration of
HgCl2 in the 10% CH3CN/HEPES buffer (10 mM, pH 7.4); (b) Time-dependent fluorescence
intensity for a 1:1 mixture of probe 1 (10 μM) and HgCl2 at various pHs.
Figure S5. Electronic density of the HOMO and LUMO states of probe 1 and product 2 calculated by a
DFT quantum mechanical method.
Table S1. Quantum mechanical calculation data.
Table S2. Fluorescence titrations under different conditions.
Figure S7. 1H NMR spectra for probe 1 and product 2.
S1

General Methods:
13
1H and C NMR spectra were measured with a Bruker DPX-300. UV absorption spectra were obtained
using a HP8453 UV/Vis spectrophotometer. Fluorescence spectra were recorded on a Photon Technical
International Fluorescence system. Quantum mechanical calculations at the DFT level were reported by
Wavefunction, Inc. Spartan '08 (Linux version) program. Commercially available reagents were used
without further purification. Anhydrous solvents for organic synthesis were prepared by solvent
purification tower. Thin-layer chromatography (TLC) was performed on precoated silica gel 60F-254
glass plates. pH-dependent fluorescence experiments and detection limit experiments were recorded by
fluorescence Victor multi-label counter with excitation wavelength at 355nm and detection wavelength at
460nm.
Synthesis of probe 1:
Reference: Goud, T, V.; Tutar, A.; Biellmann, J. Tetrahedron. 2006, 62, 5084-5091.
SMe
SMe
S
H
(b)
(a)
(c)
N
NH HN
I
N
N
B
F
NH HN
F
3
4
Probe 1
Scheme 1. (a) (i) Thiophosgene (0.48 equiv.), Toluene, Et2O, 25 °C, 10 min. (ii) 10% MeOH in water,
25 °C, 30 min. (b) CH3I (18 equiv.), DCM, 25 °C, 21 h. (c) Et3N (5.3 equiv.), BF3·Et2O (4.9 equiv.),
dichloromethane, 25 °C, 24 min.
Bis-(1H-pyrrol-2-yl)-methione (3): A solution of pyrrole (4.06 g, 4.2 mL, 59.4 mmol) in dry ether (90
mL) under argon atmosphere was added dropwise to a vigorously stirred solution of thiophosgene (3.45 g,
2.3 mL, 30.0 mmol) in dry toluene (80 mL) at 0 °C. After 10 min, aqueous methanol (10% methanol) (ca.
100 mL) was added and the mixture stirred for further 30 min at room temperature. The solvents were
removed in vacuo and the residue was chromatographed on neutral alumina (column diameter = 4 cm,
height of neutral alumina = ca 20 cm, eluent: hexane/dichloromethane = 1/3) The pure compound
fraction was collected, which, after removal of the solvents in vacuo, yielded the thioketone 3 as a
1
crystalline dark-red solid (3.86 g, 76%). H NMR (CDCl3, 300 MHz, 293K): δ 6.40 (2H, m), 7.04 (2H,
m), 7.19 (2H, m), 9.76 (2H, s); 13C NMR (CDCl3, 75 MHz, 293K): δ 112.5, 114.8, 127.7, 138.4, 193.2.
2-[Methylsulfanyl-(1H-pyrrol-2-yl)-methylene]-2H-pyrrolium iodide (4) : To a solution of compound
3 (1.10 g, 6.24 mmol) in anhydrous dichloromethane (18 mL) under argon atmosphere was added methyl
iodide (16.0 g, 7.0 ml, 112 mmol) at room temperature. The reaction mixture was stirred for 21 h (TLC
monitoring). Solvent was removed under reduced pressure to obtain a brownish and gummy solid (2.04
g). The compound 4 was used for the next reaction without further purification.
8-(Thiomethyl)4,4-difluoro-4-bora-3a,4a-diaza-sindacene (Probe 1): To a solution of compound 4
(2.04 g) in anhydrous dichloromethane (45 mL) under argon atmosphere at room temperature was added
triethylamine (3.34 g, 4.6 mL, 33.0 mmol). After being stirred for 30 min, BF3·Et2O (4.37 g, 3.8 mL, 30.8
mmol) was added to the mixture. The mixture was stirred for 24 h at room temperature. Solvent was
evaporated under vacuum, and the residue was chromatographed on silica gel (column diameter = 3 cm,
S2

height of silica gel = ca 20 cm, eluent: hexane/dichloromethane = 10/1 → 1/1 → 1/2) to yield probe 1 as
1
a crystalline dark red solid (404 mg, 26% from two steps). H NMR (CDCl3, 300 MHz, 293K): δ 7.80
(2H, m), 7.26-7.42 (2H, m), 6.50-6.51 (2H, m), 2.91 (3H, s); 13C NMR (CDCl3, 75 MHz, 293K): δ 154.0,
140.8, 133.4, 127.3, 117.6, 20.0.
3.5x106
0.4
3.0x106
0.3
2.5x106
2.0x106
0.2
1.5x106
0.1
1.0x106
5.0x105
0.0
0.0
400
450
500
550
600
300
350
400
450
500
550
600
Wavelength (nm)
Wavelength (nm)
(a)
(b)
3.5x106
6x105
5x105
4x105
3x105
2x105
3.0x106
2.5x106
2.0x106
1.5x106
1.0x106
5.0x105
0.0
1x105
0
0
30
60
90 120 150 180 210
0
30
60
90 120 150 180 210
Time (min)
Time (min)
(c)
(d)
Figure S1. Time-dependent absorbance and fluorescence emission spectra of probe 1 (10uM) upon
addition of HgCl2 (1:1eq) in the 10% CH3CN/HEPES buffer (10 mM, pH 7.4). (a) Absorbance spectra:
each spectrum was recorded after 0, 30, 50, 70, 90, and 110 min. (b) Fluorescence emission spectra: each
spectrum was recorded after 0, 10, 30, 50, 70, 90, 110, 130, 150, 170, 190, and 210 min. Excitation
wavelength = 370 nm. (c) A plot of fluorescence intensity that is estimated as the peak height at 409 nm.
(d) A plot of fluorescence intensity that is estimated as the peak height at 525 nm.
S3

0.4
0.3
0.2
0.1
0.0
300
350
400
450
500
550
600
Wavelength (nm)
(a)
(b)
Figure S2. Absorbance and fluorescence emission data for a 1:1 mixture of probe 1 (10 μM) and each of
the various metal ions (Hg2+, Ni2+, Ba2+, Cr2+, Au3+, Pb2+, Mn2+, Ag+, Co2+, Al3+, Cd2+, Fe3+, Cu2+, Pd2+,
Mg2+, Ca+, and Zn2+; as the chloride salts) in the 10% Ch3CN/HEPES buffer (10 mM, pH 7.4), acquired
after 180 min: (a) absorbance spectra; (b) fluorescence intensity at 409 nm (excitation wavelength = 370
nm).
1x103
1x103
1x103
1x103
1x103
9x102
8x102
7x102
1.2x104
1.0x104
8.0x103
6.0x103
4.0x103
2.0x103
0.0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0
0
20
40
60
80
100
[HgCl ] (ppb)
2
[HgCl ] (ppb)
2
(a)
(b)
Figure S3. A plot of fluorescence intensity depending on the concentration of HgCl2 in the range from
0.25 ppb to 100 ppb. Each measurement was done after 3 h of mixing for a probe 1 and HgCl2 in the
HEPES buffer (10 mM, pH 7.4) containing 10% CH3CN. Fluorescence intensity was recorded by Victor
multi-label counter with excitation wavelength at 355 nm and detection wavelength at 460 nm: (a) a
region between 0 ppb to 5 ppb [HgCl2]; (b) a region between 0 ppb to 100 ppb [HgCl2].
S4

3.5x102
3.0x102
2.5x102
2.0x102
1.5x102
1.0x102
3.5x106
3.0x106
2.5x106
2.0x106
1.5x106
1.0x106
pH 4
pH 6
pH 7
pH 8
pH 10
5.0x101
0.0
5.0x105
0.0
0
20 40 60 80 100120140160180200220
400
450
500
550
600
Wavelength (nm)
Time (min)
(a)
(b)
Figure S4. (a) Fluorescence spectra of probe 1 (10 μM) in the presence of different concentration of
HgCl2 (0, 0.5, 1, 3, 5, 10, 20 equiv.) in the 10% CH3CN/HEPES buffer (10 mM, pH 7.4), acquired after
60 min (black line: 0 equiv.; indigo line: 20 equiv.). (b) Time-dependent fluorescence intensity for a 1:1
mixture of probe 1 (10 μM) and HgCl2 at the various pHs (pH 4, 6, 7, 8, 10) by Victor multi-label counter
with excitation wavelength at 355 nm and detection wavelength at 460 nm.
Table S1. Quantum mechanical calculation data for probe 1, product 2 and sulfoxo-BODIPY 6.
Me
O
SMe
OH
S
B
N
N
B
F
N
N
N
N
B
F
F
F
F
F
Probe 1
Product 2
Sulfoxo-BODIPY 6
1st Ex. Energy (au) 1st Ex. Energy (eV) E LUMO (eV) E HOMO (eV)
∆ E(eV)
3.0284
3.4836
3.0062
λ nm
1
2
6
0.11637
0.13440
0.10885
3.1666
3.6571
2.9618
-2.9630
-2.5483
-3.1657
-5.9915
-6.0319
-6.1718
391.27
338.79
418.33
S5

Figure S5. Electronic density of the HOMO and LUMO states of probe 1 and product 2 obtained by DFT
quantum mechanical calculations.
Table S2. Fluorescence titrations under different conditions.
Solvent
CH3CN
Hg2+ (equiv.)
Result [c]
Condition 1
Condition 2
Condition 3
Condition 4
Condition 5
0
0
1
1
1
No ratiometric behavior
No ratiometric behavior
No ratiometric behavior
Ratiometric behavior[b]
Ratiometric behavior
HEPES buffer [a]
CH3CN
HEPES buffer
CH3CN-HEPES buffer (1:1)
[a] HEPES buffer (pH 7.4, 10 mM). [b] Ratiometric result under UV radiation. [c] General conditions:
50 μM probe 1, at 28 °C, for 6 h, under argon atmosphere.
S6

1H NMR data
8-(Thiomethyl)-4,4-difluoro-4-bora-3a,4a-diaza-sindacene (Probe 1).
8-(Hydroxy)-4,4-difluoro-4-bora-3a,4a-diaza-sindacene (product 2)
S7