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LI ET AL.
there are few reports about the application of fluorescent probe on the
detection of amino acid.28,29
Compounds 1 to 4 were synthesized according to the route shown
in Scheme 1.
Based on the above consideration, we have rationally designed and
synthesized a series of Schiff base derivative having an aldehyde group
moiety as a signaling unit and conjugated imine functionality (C N) as a
reaction unit and naphthalene as a fluorescent group (Scheme 1). Results
indicated that the synthesized compounds showed the strongest
response and high specificity for Arg among normal amino acids tested
(alanine, valine, leucine, isoleucine, methionine, aspartic acid, glutamic
acid, glycine, serine, threonine, asparagine, phenylalanine, histidine, tryp-
tophan, proline, lysine, glutamine, tyrosine, and cysteine), which accom-
panied significant fluorescent strength. Therefore, the compounds can
be used as a fluorescent probe for the detection of Arg.
Compound 1: An organic ethanol solution of 4‐
nitrophenylhydrazine (10 mmol, 1.5314 g) was added to a 3‐necked
flask, and a hot ethanol solution of 1‐naphthaldehyde (10 mmol,
1.7218 g) was added drop wise to the above solution, heating, mag-
netic stirring, so that completely dissolved, and soon, the color of the
solution changed from light yellow to brick red. The mixture reacted
at room temperature for 40 minutes and refluxed for 6 hours. Then,
cooled and stood, brick‐red solid appeared. Filtered, washed with
anhydrous ethanol, and recrystallized by dichloromethane and anhy-
drous methanol in a volume ratio of 1:1 and brick‐red solid obtained.
Yield: 62%. Proton nuclear magnetic resonance (400 MHz, DMSO) δ
11.44 (s, 1H), 8.74 (d, J = 11.0 Hz, 2H), 8.19 (d, J = 9.4 Hz, 2H), 8.08
to 7.91 (m, 3H), 7.70 (ddd, J = 8.4, 6.8, 1.4 Hz, 1H), 7.65 to 7.54 (m,
|
2H), 7.24 (d,
J = 8.7 Hz, 2H). Elemental analysis: calc for
2
MATERIAL AND METHODS
C
17H13N3O2: C, 70.09; H, 4.50; N, 14.42; found: C, 70.32; H, 4.19;
Most of the starting materials were obtained commercially, and all
reagents and solvents used were of analytical grade. All amino acids were
purchased from Aladdin Chemistry Co Ltd (Shanghai, China) and stored in
a desiccator under vacuum and used without any further purification.
Dimethyl sulfoxide (DMSO) was distilled in a vacuum after being dried
with CaH2. C, H, and N elemental analyses were made on a Vanio‐EL
instrument. Proton nuclear magnetic resonance (1H‐NMR) spectra were
recorded on a Unity Plus‐400‐MHz spectrometer. Electrospray ioniza-
tion mass spectrometry was performed with a liquid chromatography
mass spectrometry apparatus (Agilent, Palo Alto, CA, USA). Ultraviolet‐
visible (UV‐Vis) titration experiments were made on a Shimadzu
UV2550 spectrophotometer at 298 K. Fluorometric titration was per-
formed on a Cary Eclipse Fluorescence Spectrophotometer at 298 K
(Agilent, Palo Alto, CA, USA). The binding constant, Ks, was obtained by
nonlinear least square calculation method for data fitting.
N, 14.62. High‐resolution mass spectrometry (m/z): 290.0921 (M─H)−.
Compound 2: The synthesis method was similar to the above pro-
cedure. Yield: 75%. Proton nuclear magnetic resonance (400 MHz,
DMSO) δ 11.87 (s, 1H), 9.58 (s, 1H), 8.92 (d, J = 2.6 Hz, 1H), 8.68 (d,
J = 8.4 Hz, 1H), 8.45 (d, J = 12.2 Hz, 1H), 8.21 to 8.13 (m, 2H), 8.08
(dd, J = 15.8, 8.2 Hz, 2H), 7.76 to 7.60 (m, 3H). Elemental analysis: calc
for C17H12N4O4: C, 60.71; H, 3.60; N, 16.66; found: C, 60.47; H, 3.75;
N, 16.89. High‐resolution mass spectrometry (m/z): 335.0781 (M─H)−.
Compound 3: The synthesis method was similar to the above pro-
cedure. Yield: 64%. Proton nuclear magnetic resonance (400 MHz,
DMSO‐d6, 298 K) δ 11.42 (s, 1H), 8.99 (s, 1H), 8.77 (d, J = 8.6 Hz),
8.20 (d, J = 9.3 Hz), 7.88 (d, J = 8.7 Hz), 7.69 to 7.52 (m), 7.39 (dd,
J = 21.6, 14.5 Hz), 7.24 (d, J = 8.9 Hz), 7.12 (d, J = 9.0 Hz). Elemental
analysis: calc for C17H13N3O3: C, 66.44; H, 4.26; N, 13.67; found: C,
66.50; H, 4.19; N, 13.93. High‐resolution mass spectrometry (m/z):
306.0887 (M─H)−.
The cells at logarithmic growth phase were seeded in a 96‐well
plate at a density of 2.0 × 104 cells/well and cultured for 24 hours.
After that, the culture media were replaced with 200 μL of Roswell
Park Memorial Institute 1640 medium containing different concentra-
tions of the compound, and the cells were further incubated for
24 hours. Next, the cells were washed with phosphate‐buffered saline
3 times, and then 100 μL of culture medium and 20 μL of MTT solution
were respectively added to each well. After the additional incubation
(4 hours), the absorbance of each well was detected at 490 nm by
using the microplate reader (Thermo Multiscan MK3, Thermo Fisher
Scientific, MA, USA) with the plain cell culture media as the control.
The survival curves were plotted, and the IC50, defined as the com-
pound concentrations required for 80% inhibition of cell growth, was
calculated based on the survival curves.
Compound 4: The synthesis method was similar to the above pro-
cedure. Yield: 80%. Proton nuclear magnetic resonance (400 MHz,
DMSO‐d6, 298 K) δ 12.23 (s, 1H), 9.33 (s, 1H), 9.23 (d, J = 8.5 Hz),
8.89 (dd, J = 9.6, 2.7 Hz), 8.35 (dd, J = 25.1, 9.2 Hz), 8.15 to 8.00 (m),
7.85 (t, J = 7.5 Hz), 7.68 (d, J = 8.9 Hz). Elemental analysis: calc for
C17H12N4O5: C, 57.96; H, 3.43; N, 15.90; found: C, 58.04; H, 3.51;
N, 15.77. High‐resolution mass spectrometry (m/z): 351.0769 (M─H)−.
|
3
RESULTS AND DISCUSSION
|
3.1
Ultraviolet‐visible titration
The binding abilities of synthesized compounds with amino acids were
investigated by using UV‐Vis absorption method in DMSO‐H2O (1:1,
v/v) at 298 K. The UV‐Vis spectral changes of the compounds were
shown in Figure 1 during the titration with Arg. In the absence of
Arg, compound 4 (4.0 × 10−5 mol L−1 in DMSO) exhibited an obvious
peak at 420 nm. With the increase of Arg, the intensity of absorption
peak at 420 nm decreased remarkably and a new absorption peak
appeared centered at about 510 nm. As a result, the absorption peak
shifted to the long wavelength direction gradually and red‐shift
SCHEME 1 Synthesis route for compounds