J. Kong et al. / Dyes and Pigments 101 (2014) 254e260
255
Table 1
2. Experiment
Crystal data and structure refinement for RBS.
2.1. Materials and methods
Empirical formula
Formula weight
Crystal size
C37 H39.50 N5 O3.25
606.24
0.24 ꢀ 0.26 ꢀ 0.28 mm3
All the reagents and solvents unless otherwise stated, were
purchased from commercial sources and used without further
purification. Adenosine-50-triphosphate disodium trihydrate (ATP),
Adenosine-50-diphosphate disodium (ADP), Adenosine-50-mono-
phosphate acid monodydrate (AMP), Cytidine-50-triphosphate
disodium dihydrate (CTP), Cytidine-50-diphosphate disodium hy-
drate (CDP), Cytidine-50-diphosphate acid (CMP), Guanosine-50-
triphosphate disodium hydrate (GTP), Guanosine-50-diphosphate
disodium (GDP), Guanosine-50-monophosphate disodium (GMP),
Uridine-50-triphosphate trisodium (UTP), Uridine-50-diphosphate
disodium (UDP) and Uridine-50-monophosphate disodium (UMP)
were purchased from Bio Basic Inc. (BBI) company. 1H NMR spectra
were measured on a VARIAN INOVA-400 spectrometer with
chemical shifts reported as ppm (in d6-DMSO, CDCl3 or D2O, TMS as
internal standard). ESI mass spectra were carried out on an HPLC-Q-
Tof MS spectrometer. The solution fluorescent spectra were
measured on EDINBURGH FS920. Optical absorption spectra were
measured on a TU-1900 UveVis spectrophotometer at room tem-
perature. The fluorescence imaging for intracellular nucleoside
polyphosphates in HeLa cells was observed under Nikon eclipse
TE2000-5 inverted fluorescence microscopy with a 20ꢀ objective
lens (excited with green light) and Olympus FV1000 laser scanning
microscopy with a 40ꢀ objective lens (excited with 510 nm).
Single crystals data of compound RBS was collected on a
Temperature (K)
293(2)
0.71073 A
ꢀ
ꢀ
Wavelength (A)
Crystal system
Space group
Monoclinic
C2/c
ꢀ
a(A)
24.7312(12)
25.7108(12)
20.9872(12)
91.845(4)
13,338.0(12)
16
1.208
0.078
5160
1.86e25.00 deg.
34,605
ꢀ
b(A)
ꢀ
c(A)
b
(deg)
3
ꢀ
V(A )
Z
Dc(g cmꢁ3
)
m
(mmꢁ1
)
F(000)
Theta range for data collection
Reflections collected
Independent reflections
Completeness to theta ¼ 25.00
Absorption correction
11,668 (Rint ¼ 0.0470)
99.2%
None
Refinement method
Full-matrix least-squares on F2
Goodness-of-fit on F2
1.031
Final Rindices [I > 2
Rindices (all data)
Largest peak and hole (e A
CCDC number
s
(I)]
R1 ¼ 0.0845a, wR2 ¼ 0.2178a
R1 ¼ 0.2180a, wR2 ¼ 0.2546a
0.453 and ꢁ0.277
954044
ꢀꢁ3
)
P
P
P
P
a
R1
¼
ðjF0j ꢁ j:Fcj:Þ= j:F0j: ; wR2 ¼ ½ wðjF0j ꢁ j:Fcj:Þ2= wF02ꢂ1=2
:
Reactions were monitored by TLC. After cooling, neutralized with
NH4OH (15%) and sodium hydroxide until pH ¼ 8, extracted with
CHCl3 for several times, while the extraction solution was moni-
tored, washed with brine, dried over anhydrous magnesium sulfate,
and concentrated to yield a dark-red solid which was recrystallized
from toluene to afford 2.30 g of the products (52%); 1H NMR (CDCl3,
ppm): 7.82 (d, 1H, J ¼ 4.0 Hz), 7.80 (d, 1H, J ¼ 4.0 Hz), 7.07 (d, 1H,
J ¼ 8.0 Hz), 6.71 (d, 1H, J ¼ 8.4 Hz), 5.08 (s, 2H), 2.69 (s, 3H).
BRUKER SMART APEXCCD diffractometer with graphite-
ꢀ
monochromated Mo-K
a
(l
¼ 0.71073 A) using the SMART and
SAINT programs. The skeleton non-hydrogen atoms were refined
anisotropically and hydrogen atoms within the ligand backbones
were fixed geometrically at calculated distances and allowed to ride
on the parent non-hydrogen atoms.
Cell imaging was measured on Nikon eclipse TE2000-5 inverted
fluorescence microscopy and Olympus FV1000 laser scanning mi-
croscopy. HeLa cells were cultured in 1640 supplemented with 10%
FCS (Invitrogen). Cells were seeded on 18 mm glass cover slips for
fluorescence imaging and in 24-well flat-bottomed plates. After
2.3. Synthesis of RBS [16e18]
To a solution of rhodamine B (1.0 g, 2.1 mmol) in dry 1,2-
dichloroethane (15 mL) under stirring phosphorus oxychloride
(1.9 mL, 21 mmol) was added dropwise over a period of 10 min,
with a pressure-equalizing addition funnel under Ar. After being
heated to reflux for 4 h, the solvent and excess amount of phos-
phorus oxychloride was removed by rotary evaporation to give the
corresponding acid chloride, which was dried over high vacuum
and used for the next step without further purification. To a solu-
tion of the acid chloride in dry acetonitrile (5.0 mL) was added
dropwise a solution of AMND (0.5 g, 3.15 mmol) and triethylamine
(5 mL) in dry acetonitrile (10.0 mL), the resulting mixture was
heated to reflux for 5 h. The reaction mixture was then concen-
trated under vacuum, and the residue was purified by column
chromatography (ethyl acetate/dichloromethane 1/1, v/v) to give
the crude product, which was further purified by recrystallization
from dichloromethane/hexane (25/1, v/v) to give a pure compound
RBS as a white solid in 22% yield (0.275 g); 1H NMR (d6-DMSO,
ppm): 1.03 (12H, m), 2.57 (3H, s), 3.24e3.34 (8H, m), 6.15 (1H, d,
J ¼ 2.6 Hz), 6.17 (1H, d, 2.6, J ¼ 8.8 Hz), 6.40 (4H, m), 7.05 (1H, d,
J ¼ 8.8 Hz), 7.32 (1H, d, J ¼ 7.7 Hz), 7.59 (1H, t, 8.8 Hz), 7.64 (1H, t,
J ¼ 7.7 Hz), 7.97 (1H, d, J ¼ 7.2 Hz), 8.12 (1H, d, J ¼ 8.0 Hz), 8.25 (1H,
d, J ¼ 8.8 Hz), 8.47 (1H, d, J ¼ 8.8 Hz); 13C NMR 168.67, 162.27,
154.63, 154.20, 153.52, 152.57, 148.50, 137.32, 135.75, 133.73, 129.90,
128.12, 128.05, 124.30, 123.41, 121.35, 118.25, 116.04, 107.98, 107.40,
12 h, HeLa cells were incubated with 10 mM compound RBS buffer
solution (pH ¼ 6.04) for 30 min at room temperature and then
washed with physiological brine three times before incubating
with 40 eq nucleoside polyphosphates (pH ¼ 6.04) for another
30 min, and cells were rinsed with physiological brine three times
again. The fluorescence imaging of intracellular nucleoside poly-
phosphates in HeLa cells was observed under Nikon eclipse
TE2000-5 inverted fluorescence microscopy with a 20ꢀ objective
lens (excited with green light) and Olympus FV1000 laser scanning
microscopy with a 40ꢀ objective lens (excited with 510 nm).
The binding constant was calculated from the fluorescent
titration curve according to the equation.
LogððF ꢁ FminÞ=ðFmax ꢁ FÞÞ ¼ log k þ n log½cꢂ
where F is fluorescent intensity of RBS at 583 nm upon the addition
of different amount of nucleoside polyphosphates. [c] stands for the
concentration of nucleoside polyphosphates.
2.2. Synthesis of 2-amino-7-methyl-1,8-naphthyridine (AMND)
[14,15]
2,6-Diaminopyridine (3.00 g, 27.5 mmol) was dissolved in 35 mL
of H3PO4 at 90 ꢃC under Ar atmosphere, 3-ketobutanal dimethyl
acetal (3.70 g, 28.2 mmol) was slowly added from a pressure-
equalizing addition funnel, and the mixture was heated at 115 ꢃC.
98.01, 66.85, 44.26, 25.60, 12.61; TOF-ESI-MS: Calcd for [M þ 2H]2þ
:
292.6552 m/z. Found: m/z 292.7144; Calcd for [M þ H]þ: m/z
584.3026. Found: m/z 584.4264.