M.-Q. Wang et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
well as to modulate the cellular uptake. However, the effect on the
substitutions on the quinoline nitrogen atom for detection of G-quad-
ruplex is rarely found in literature. Upon further investigation of this
series, in the present study, we designed three N-alkylated styr-
ylquinolinium dyes Ls-1, Ls-2 and Ls-3 with different groups at the
chain end. Their photophysical characterization and fluorescence per-
formance on various DNA forms were investigated. These dyes were
able to bind with nucleic acids, and dye Ls-2 with a sulfonato group at
the chain end displayed excellent fluorescent signal discrimination to G-
quadruplex DNA. The detailed binding properties for G-quadruplex
DNA were assessed through both experimental and modeling studies. In
addition, the intracellular localization and cytotoxicity were also ex-
plored.
(positive mode, m/z) calculated 466.2164, found 466.2162 for [M
+
+H]
.
2.2.3. Characterization of Ls-3
1
Yield 67.3%, H NMR (400 MHz, DMSO‑d ) δ: 9.19 (d, J = 6.68 Hz,
6
1H), 9.06 (d, J = 8.16 Hz, 1H), 8.39–8.36 (m, 2H), 8.25–21 (m, 1H),
8.18–8.14 (m, 1H), 8.11–8.07 (m, 1H), 8.02 (t, J = 7.52 Hz, 1H), 7.89
(d, J = 8.96 Hz, 2H), 7.07 (d, J = 8.96 Hz, 2H), 4.47 (s, 3H), 3.38–3.36
1
3
(m, 4H), 2.49–2.47 (m, 4H), 2.24 (s, 3H);
C NMR (100 MHz,
DMSO‑d ) δ: 153.48, 152.42, 147.71, 144.19, 139.23, 135.23, 131.40,
6
129.28, 126.93, 126.42, 126.17, 119.64, 115.62, 115.25, 115.00,
53.81, 46.09, 44.82, 44.59, 44.24, 22.67, 22.05; HRMS: (positive mode,
m/z) calculated 344.2121, found 344.2083 for [M-I]+
;
2
. Experimental methods
.1. Materials and methods
-Methyl-1-(4-sulfobutyl)quinolinium inner salt,
2.3. Measurements and methodology
2
2.3.1. Spectrophotometric studies
The absorption spectra were recorded on an UV-2550 spectro-
photometer using a 1 cm path length quartz cuvette at room tempera-
ture. For the titration experiment, small aliquot of a stock solution of
DNA was added to the solution containing the dye at a fixed con-
centration (8 μM) in aqueous buffer (10 mM Tris-HCl, pH 7.4, 60 mM
KCl). After each DNA addition, the solution was incubated for 2 min
before absorption spectra recorded.
2
4
4
1-(3-hydro-
and 1,4-dimethyl-quinoli-
2
5
xypropyl)-4-methyl-quinolinium iodide
1
3
nium iodide were synthesized according to previously described lit-
erature protocols. All commercially available chemicals, solvents, used
for synthesis, were reagent grade and used without further purification.
All oligonucleotides were purchased from Sangon Biotechnology Co.,
Ltd. (Shanghai, China) and their sequences were listed in Table S1. All
the oligonucleotides were dissolved in Tris − HCl buffer (10 mM, con-
taining 60 mM KCl, pH 7.4). Stock solutions of Ls-1, Ls-2 and Ls-3 were
2.3.2. Fluorimetric titrations
Fluorescence spectra were measured on a Shimadzu RF-5301PCS
spectrofluorophotometer in a 10 mm quartz cell at room temperature.
The concentration of the dye was fixed at 2 μM and the titration process
was similar to absorption spectra titration experiment. The date from
the fluorimetric titrations were analyzed according to the independent-
1
13
prepared in DMSO. H NMR and C NMR spectra were measured on a
1
13
4
00 MHz spectrometer (400 MHz for H and 100 MHz for C) using
TMS as internal standard in DMSO‑d . High resolution mass spectra
HRMS) were recorded on a Shimazu LCMS-IT-TOF instrument with an
ESI detector.
6
(
2
6
site model by nonlinear fitting to Eq. (1), in which F is the fluores-
0
cence intensity of the dye in the absence of G-quadruplex DNA, n is the
2.2. General method for preparation of quinolinium derivatives
putative number of the dye binding to a given DNA matrix, Q is the
fluorescence enhancement upon saturation, A = 1/[K
a
C
dye
]
and
4
-(4-Methylpiperazino)benzaldehyde (1.1 mmol) and quinolinium
x = nC
/C . The parameters Q and A were found by Levenberg-
DNA dye
salts (1.0 mmol) were added to a 100 mL flask with 40 mL anhydrous
ethanol, followed by 5 drops catalytic piperidine. The resulting mixture
was allowed to reflux for 15 h under nitrogen with stirring. After
cooling to room temperature, the solvent was removed under reduced
pressure and the residue was purified by column chromatography on
Marquardt fitting routine in Origin 8.5 software.
F
F0
Q
1
2
4x]
2
(1)
silica gel eluting with CH
as a brownish solid.
2
Cl
2
/CH
3
OH to afford quinolinium derivatives
2.3.3. Circular dichroism (CD)
CD spectra (230–500 nm) were performed on a JASCO-J815 circular
dichroism spectrophotometer using a 10 mm path length quartz cuvette
at 25 °C. The scanning speed of the instrument was set to
2.2.1. Characterization of Ls-1
1
−1
Yield 43.5%, H NMR (400 MHz, DMSO‑d
6
) δ: 9.34 (d, J = 6.56 Hz,
500 nm min . The strand concentration of G-quadruplex used for
1
H), 9.06 (d, J = 8.60 Hz, 1H), 8.48 (d, J = 8.96 Hz, 1H), 8.40 (d,
measurement was 4 μM. CD signals were recorded with a band width of
1 nm and final analysis of the data was carried out using Origin 8.5.
J = 6.68 Hz, 1H), 8.20–8.16 (m, 2H), 8.08–8.04 (m, 1H), 7.96 (t,
J = 8.08 Hz, 1H), 7.88 (d, J = 8.72 Hz, 2H), 7.03 (d, J = 8.80 Hz, 2H),
5
3
1
1
5
.13 (s, 1H), 5.02 (t, J = 6.92 Hz, 2H), 3.52–3.37 (br, 10H), 2.26 (s,
2.3.4. Molecular docking
1
3
H), 2.10–2.07 (m, 2H); C NMR (100 MHz, DMSO‑d
6
) δ: 153.68,
Molecular docking calculations were performed using the Autodock
Vina software, which has been reported to be of high accuracy of pre-
52.92, 147.41, 144.63, 138.31, 135.25, 131.52, 129.05, 127.28,
26.76, 125.74, 119.40, 115.29, 115.19, 114.66, 60.21, 57.83, 54.50,
2
7
and the NMR structure (PDB code: 143D) of G-quadruplex DNA were
downloaded from RCSB Protein Data Bank. The redundant solvent
molecules and ions were removed from the crystal structure while the
first conformation was retained from the NMR structure. Docked poses
were visualised by using UCSF Chimera.
4.27, 21.24, 14.56; HRMS: (positive mode, m/z) calculated 388.2383,
+
found 388.2349 for [M-I]
;
2.2.2. Characterization of Ls-2
1
Yield 51.1%, H NMR (400 MHz, DMSO‑d
6
) δ: 9.24 (d, J = 6.72 Hz,
1
H), 9.04 (d, J = 8.08 Hz, 1H), 8.54 (d, J = 8.92 Hz, 1H), 8.39 (d,
J = 6.72 Hz, 1H), 8.20–8.14 (m, 2H), 8.09–8.05 (m, 1H), 7.98 (t,
J = 7.76 Hz, 1H), 7.88 (d, J = 8.96 Hz, 2H), 7.06 (d, J = 8.96 Hz, 2H),
2.3.5. Fluorescence intercalator displacement (FID) assays
FID assays were also performed on a Shimadzu RF-5301PCS spec-
trofluorophotometer. The experiments were carried out in 10 mM Tris-
HCl buffer (pH 7.4, containing 60 mM KCl) by adding different con-
centrations of dyes to the TO-bound G-quadruplex solution. The con-
centrations of TO and G-quadruplex were set at 0.5 and 0.25 μM, re-
spectively. The fluorescence spectra were measured using excitation
4
.94 (t, J = 7.24 Hz, 2H), 3.38–3.33 (m, 4H), 2.49–2.44 (m, 6H), 2.23
13
(
s, 3H), 2.05–1.99 (m, 2H), 1.73–1.65 (m, 2H); C NMR (100 MHz,
DMSO‑d ) δ: 153.69, 153.01, 147.02, 144.65, 138.33, 135.31, 131.50,
29.12, 127.14, 126.76, 125.68, 119.58, 115.25, 114.65, 56.45, 54.66,
2.71, 51.04, 50.89, 46.94, 45.96, 28.91, 23.31, 22.98, 22.64; HRMS:
6
1
5
2