B
C. Huang et al.
S
S
dropwise. The reaction mixture was stirred for 1 h at 08C, and
then for 12 h at room temperature, followed by the removal of
THF under reduced pressure. Water was added to the reaction
mixture, and the product was extracted with dichloromethane
(4 ꢀ 10 mL). The organic layer was dried with dry Na2SO4,
followed by evaporation of the solvent. The crude product was
separated by column chromatography with a gradient of hexane
in dichloromethane (20–0 %) and ethyl acetate in dichloro-
methane (0–20 %). The resulting solid was recrystallised from
acetone to give compound 8 (453 mg, 65 %) as a yellow powder.
nmax (KBr)/cmꢁ1 2223 (CꢃN) and 1594–1348 (C¼C). dH
([D6]DMSO, 400 MHz) 8.446 (2H, s, Ph), 7.620 (2H, d, J 16.4,
CH=CH), 7.498 (4H, d, J 8.4, Ph), 7.054 (2H, d, J 16.4,
CH=CH), 6.844 (4H, d, J 8.8, Ph), 3.772 (8H, t, J1 ¼ J2
4.4 Hz, NCH2), 3.712 (8H, t, J1 ¼ J2 2 Hz, CH2Cl). Found: C
62.80, H, 4.98, Cl 23.13, N 9.10 %; [M]þ 610.1225. Anal. Calc.
for C32H30Cl4N4 (612.42) C 62.76, H 4.94, Cl 23.16, N 9.15 %;
[M]þ 610.1225.
CN
OH
OH
S
S
N
HO
HO
N
NC
BHg
Fig. 1. Molecular structure of probe BHg.
(HSA) as a novel Hg2þ ligand.[12] Although this probe exhibited
excellent selectivity for Hg2þ, it was not applied to live cell and
living tissues imaging.
Herein, we extend our earlier work[12] and report a new TPEF
probe for Hg2þ derived from bis(styryl)terephthalonitrile as a
two-photon fluorophore and bis[2-(2-hydroxyethyl sulfanyl)
ethyl]amino group (ionophore) as a novel Hg2þ ligand. The
probe, 2,5-bis((E)-4-(bis(2-(2-hydroxyethylthio)ethyl)amino)
styryl)terephthalonitrile (BHg), contains two sulfur atoms
known as ‘soft base’ capable of chelating so-called ‘soft acid’
heavy metal cations and exhibits good affinity for Hg2þ. We
report that BHg (Fig. 1) is capable of imaging Hg2þ ions in live
cells without mistargeting and photobleaching problems.
2,5-Bis((E)-4-(bis(2-(2-hydroxyethylthio)ethyl)amino)
styryl)terephthalonitrile (BHg)
Compound
8 (306 mg, 0.5 mmol), 2-mercaptoethanol
Experimental
(172 mg, 2.2 mmol), and anhydrous K2CO3 (414 mg, 3 mmol)
were dissolved in acetone (25 mL). Then, the mixture was
refluxed for 24 h with stirring under N2. The resulting mixture
was filtered, and the filtrate was concentrated by evaporating the
solvent to obtain a viscous liquid. The crude product was purified
by column chromatography using acetone/dichloromethane to
afford compound BHg (335mg, 86%) as a red solid. Further
purification could be achieved by recrystallisation from methanol
to give needles.
nmax (KBr)/cmꢁ1 3422 (OH), 2922 (CH), 2220 (CꢃN),
1631–1349 (C¼C). dH ([D]CHCl3, 400MHz) 8.442 (2H, s, Ph),
7.620 (2H, d, J 16.0, CH¼CH), 7.513 (4H, d, J 8.8, Ph),
7.055 (2H, d, J 16.0, CH¼CH), 6.789 (4H, d, J 8.4, Ph), 4.918
(8H, t, J 4.8, 4 ꢀ OCH2), 3.633 (8H, t, J1 ¼ J2 6.0, 4 ꢀ NCH2),
2.791 (8H, t, J1 6.8, J2 7.6, 4 ꢀ SCH2), 2.728 (8H, t, J1 6.8, J2 6.4,
4 ꢀ SCH2), 2.564 (4H, s, 4 ꢀ OH). dC ([D]CHCl3, 100 MHz)
147.68, 138.02, 134.96, 129.36, 128.88, 123.48, 117.03, 116.12,
113.15, 111.64, 61.23, 50.69, 34.11, 28.69. Found: C 61.71, H
6.54, N 7.16, O 8.17, S 16.42 %; [M]þ 778.2715. Anal. Calc. for
C40H50N4O4S4 (778.2715) C 61.66, H 6.47, N 7.19, O 8.21,
S 16.46 %; [M]þ 778.2715.
Materials and Methods
NMR spectra were recorded on a VARIAN INOVA 400 MHz
NMR spectrometer. Mass spectral determinations were made on
a electrospray ionisation quadrupole time-of-flight mass spec-
trometer (Micromass, UK). High-resolution mass spectrometry
(HRMS) was performed on a gas chromatography time-of-flight
mass spectrometer (Micromass, UK). Fluorescence measure-
ments were performed on a PTI-C-700 Felix and Time-Master
system. Fluorescence quantum yields (F) were measured using
standard methods[35] on air-equilibrated samples at room tem-
perature. Quinine bisulfate in 0.05 M H2SO4 (F ¼ 0.546) was
used as a reference.[35] TPEF action cross-section spectra were
recorded according to the experimental protocol established by
Xu and Webb[36] using a mode-locked Ti/sapphire laser that
delivers ,80 fs pulses at 76 MHz. Fluorescein (10ꢁ4 M in 0.1 M
NaOH), whose TPEF action cross-sections are well known,[36]
served as the reference. The quadratic dependence of the fluo-
rescence intensity on the excitation intensity was verified for
each data point, indicating that the measurements were carried
out in intensity regimes in which saturation or photodegradation
does not occur. The measurements were performed at room
temperature on air-equilibrated solutions (10ꢁ5 M). The
experimental uncertainty on the absolute action cross-sections
determined by this method has been estimated to be ꢂ 20 %.[36]
Absorption spectra were measured on a HP-8453 spectropho-
tometer. Solvents were generally dried and distilled before use.
Reactions were monitored by thin layer chromatography on
Merck silica gel 60 F254 pre-coated aluminium sheets. Column
chromatography was performed using Merck silica gel Si 60
(40–63 mm, 230–400 mesh). The pH-dependent fluorescence
studies were performed according to the literature.[37]
Results and Discussion
Design and Synthesis of BHg
2,5-Dibromo-p-xylene (4),[38] 2,5-dimethyl-terephthalonitrile
(5),[38] 2,5-bis(bromomethyl)terephthalonitrile (6),[38] 1,4-bis
(diethylphosphorylmethyl)-2,5-dicyano-benzene (7),[39] and
4-[bis(2-chloro-ethyl)amino]benzaldehyde (2)[40] were synthe-
sised according to literature procedures. The nucleophilic sub-
stitution of 8 and 2-mercaptoethanol gave BHg in high yield
(86 %) (Scheme 1). In the reaction of 8 and 2-mercaptoethanol,
the substitution of the mercapto group, rather than the hydroxy
group, for chloro group was observed because the nucleophilic
strength of the mercapto group is superior to that of the hydroxy
group.
Synthesis
2,5-Bis((E)-4-(bis(2-chloroethyl)amino)styryl)
terephthalonitrile (8)
Selectivity of Sensor BHg for Metal Ions
Aldehyde
2 (560 mg, 2.28 mmol) and NaH (55 mg,
2.28 mmol) were dissolved in tetrahydrofuran (THF; 3 mL),
and the solution was cooled to 08C under N2. To this solution,
phosphonate 7 (488 mg,1.14 mmol) in THF (9 mL) was added
The solubility of BHg in water was 576 mM, which is sufficient
to stain the cells (Fig. S1, available as Supplementary Material).
To obtain insights into the binding properties of BHg towards