C. Cui et al.
Inorganica Chimica Acta 520 (2021) 120285
was reacted in an ice bath for 30 min, and then reacted in room tem-
perature for 10 h. The solvent was dried by vacuum and purified by
1
column chromatography(CH2Cl2:CH3OH = 20:1) to obtain RB-AQ. H
NMR (400 MHz, CDCl3) δ 8.18 (dd, J = 6.9, 3.9 Hz, 2H), 8.10 – 8.07 (m,
1H), 7.89 (dd, J = 8.6, 4.5 Hz, 2H), 7.85 (d, J = 8.2 Hz, 1H), 7.80 (d, J =
2.3 Hz, 1H), 7.67 (dd, J = 8.2, 7.1 Hz, 1H), 7.61 (dd, J = 8.1, 7.1 Hz,
1H), 7.57 – 7.51 (m, 2H), 7.36 (dd, J = 8.9, 2.3 Hz, 1H), 7.22 – 7.19 (m,
1H), 6.76 (d, J = 8.8 Hz, 2H), 6.37 (dd, J = 8.9, 2.6 Hz, 2H), 6.32 (d, J =
2.5 Hz, 2H), 3.38 – 3.25 (m, 8H), 1.14 (t, J = 7.0 Hz, 12H). 13C NMR
(101 MHz, CDCl3) δ 168.12, 153.79, 153.74, 153.52, 153.15, 153.10,
148.93, 141.26, 139.59, 137.96, 136.17, 133.22, 131.69, 131.64,
130.37, 129.78, 129.40, 129.29, 129.24, 128.79, 128.46, 128.26,
128.07, 126.86, 124.05, 123.57, 121.67, 121.53, 108.46, 106.32, 97.99,
67.90, 44.35, 12.61. ESI-MS m/z: [M + H]+ calculated for 694.3177,
found 694.3181.
2.5. General spectrophotometric experiments
Fig. 1. Crystal structure of RB-AQ ((Hydrogen atoms were omitted for clarity,
the C, N, and O atoms were drawn in gray, blue, and red).
The metal salts (Ca(ClO4)2⋅4H2O, Cd(ClO4)2⋅6H2O, Co(ClO4)2⋅6H2O,
Mn(ClO4)2⋅6H2O, NaClO4⋅H2O, Ni(ClO4)2⋅6H2O, AgClO4⋅H2O, Cu
(ClO4)2⋅6H2O, Fe(ClO4)2⋅6H2O, Pb(ClO4)2⋅3H2O, Zn(ClO4)2⋅6H2O, Hg
(ClO4)2⋅3H2O, KClO4) were dissolved with water to prepare 1.0 × 10ꢀ 2
mol/L aqueous solution. A certain amount of RB-AQ solid was weighed
and fixed in a 10 ml volumetric flask with DMF as solvent to prepare a
1.0 × 10ꢀ 3 mol/L RB-AQ solution. The fluorescent probe RB-AQ is
configured in C2H5OH/HEPES (1:1, v/v) system (HEPES 2 mM) to
obtain a 2 µM solution. The mercury(II) ion solution of the corre-
sponding concentration was added to the probe solution for fluorescence
spectroscopy test (λex = 520 nm), and the selectivity and stability of the
probe to mercury(II) ions were recognized under the same conditions.
2.6. Crystal structure determination
The X-ray diffraction data for the fluorescent probe (RB-AQ) was
collected on a Bruker APEX-II CCD diffractometer equipped with
graphite-monochromated Mo K
α radiation (λ = 0. 71073 Å). The
structure of the probe (RB-AQ) was measured by direct methods and
refined on F2 by full-matrix least squares using SHELXTL version 6.1.
Anisotropic thermal parameters were assigned to all non-hydrogen
atoms. All hydrogen atoms were positioned geometrically and refined
using a riding model. CCDC No. 2017638 contains the crystallographic
data of the probe of this paper. The crystallographic data and details of
refinements for probe (RB-AQ) are summarized in Table S1 and Fig. S10.
The crystal data for RB-AQ: C46H39N5O2, M = 693.82, Monoclinic, P(2)
Fig. 2. Fluorescence emission spectra of RB-AQ (20
μ
M, λex = 520 nm) with an
increasing amounts of Hg2+ ions (0–500
μM) in C2H5OH/HEPES (1:1, v/v).
After that, these petri dishes were washed with PBS for three times, and
the imaging effect of RB-AQ toward Hg2+ ions in zebrafish was recorded
by laser confocal microscope. Zebrafish images were also mesured by
Olympus FV1000 laser confocal microscope with irradiating at 515 nm
and collecting emission wavelength between 550 and 650 nm.
1/c, a = 18.850(3) Å, b = 8.9064(13) Å, c = 22.598(3) Å,
α
= 90.00◦, β
= 107.748(3)◦, γ = 90.00◦, V = 3613.4(9) Å3, Z = 4, Dc = 1.275 g/cm3,
μ
= 0.079 mmꢀ 1. 32,352 reflections were collected (Rint = 0.0684). The
final refinement gave R1 = 0.0894 and wR2 = 0.1991 with I > 2sigma(I).
3. Results and discussion
2.7. Cell culture and imaging
3.1. Synthesis and characterization
The Hela cells were cultured in Dulbecco’s modified Eagle’s medium
RB-AQ was synthesized by rhodamine B and acenaphtho[1,2-b]qui-
noxaline-9-amine for 4 steps, and synthesis route was shown in Scheme
2. RB-AQ was dissolved in a mixed solution of methanol/dichloro-
methane and evaporated in room temperature to obtain single crystal of
RB-AQ. The desired structure of RB-AQ was defined by NMR spectra
(Figs. S7 and S8), ESI-MS (Fig. S9) and crystallographic data (Table S1,
Fig. S10). The crystallographic data and details of refinements for probe
(RB-AQ) are summarized in Table S1 and X-ray diffraction single crystal
structure analysis showed that RB-AQ crystallizes in the monoclinic
system P(2)1/c space group, as shown in Fig. 1. The angle of C(1)-N(1)-C
(29) and C(29)-N(1)-C(8) are 123.5◦ and 121.7◦, respectively, as shown
in Fig. S10. RB-AQ displayed a spirocycle-closed form and the spi-
rocyclic structure was almost vertical with the adjacent conjugate aro-
matic groups with the dihedral angle of about 88.8◦. The dihedral angle
(DMEM) with 10% foetal bovine serum under 5% CO2 at 37 ◦C. In order
to image Hg2+ ions in living cells, the cells were treated with 1
μM RB-
AQ in growth medium and then treated with various concentrations of
Hg2+ ions (0, 5, 15, 25
μM). After that, cells were washed 3 times with
PBS. Then cell images were obtained from Olympus FV1000 laser
confocal microscope irradiated at 515 nm and the emission wavelength
between 550 and 650 nm were collected by the red channel.
2.8. Fluorescence imaging in zebrafish
Zebrafish were selected as model organism to explore the imaging
capability of RB-AQ toward Hg2+ ions in organisms. RB-AQ (1
μM) was
added to the petri dishes containing zebrafish, and then 0, 5, 15, 25 μM
of mercury(II) ions were added to the dishes and incubated respectively.
3