Organic Letters
Letter
binding energies at 933.1 and 955.0 eV were assigned to Cu
2p3/2 and Cu 2p1/2 of Cu+, respectively.22,23
The excitation wavelength and maximum emission wave-
length of products, the interaction time, the conversion yield
through the HPLC technique, and the fluorescence intensity
enhancement of the maximum emission wavelength of S1−
S10 after treatment with the copper-cyanide complex are
shown in Table S2. Compared with S6−S10, S1−S5
substituted with p-methoxy produce products with a larger
Stokes shift. The reason may be that the conjugation of
methoxy and the benzene ring increases the π electron cloud
density and emission wavelength. In addition, S1 and S6
substituted with tert-butyl show high conversion efficiency
(more than 90%) and fast response time (10 min). The
electron-donating property of the t-butyl group not only
improves the nucleophilic addition cyclization ability of
phenolic hydroxyl groups but also facilitates subsequent
oxidation. In contrast, the conversion efficiency would be
lower as the electron-withdrawing group, e.g., bromine, was
introduced, which can easily be found from S2 and S7
compounds. Moreover, their fluorescence intensity enhance-
ment is small (less than 30-fold) because of the heavy atom
quenching effect of bromine. Compared with S2, S4, and S5
which finally form benzoxazole, benzothiazole, and benzimi-
dazole, respectively, the conversion yield to produce
benzoxazole is low (about 30%) due to its weak nucleophilic
capability of the phenolic hydroxyl group. These experimental
results further corroborate the mechanism of cyclization and
oxidation.
Considering the high conversion efficiency, fast response
time, and large fluorescence intensity enhancement (400-fold)
of S1, we take S1 as the model molecule to study fluorescent
detection of the copper-cyanide complex. In the CH3CN/PBS
(v/v = 2:1, 10 mM, pH 7.4) buffer system containing 20 equiv
of CN−, S1 is nonfluorescent upon excitation at 375 nm. As
Cu2+ was added, the fluorescence intensity at 439 nm gradually
increased. After adding 8 equiv of copper ions, a 400-fold
fluorescence enhancement was found. The fluorescence
intensity showed a good linear relationship to the concen-
tration of Cu2+ in the range 1−6 μM (Figure S7), and the
detection limit for Cu2+ is 1.53 nM. The selective and
competitive experiments of other cations for the detection of
copper ions were performed. As shown in Figure S8, the
fluorescence intensity at 439 nm increased after addition of
Cu2+, while no fluorescence was observed as other cations were
added. The fluorescence intensity increased significantly upon
addition of 5 equiv of Cu2+ in the presence of 10 equiv of
competitive cations. These results indicate that S1 in the
presence of CN− can act as a fluorescent sensor to selectively
detect Cu2+ without any interference from other analytes.
In the presence of 8 equiv of Cu2+, the fluorescence of S1
solution gradually increased as 1−30 equiv of CN− was added.
There is an obvious linear relationship between fluorescence
intensity and CN− concentration (Figure S10), demonstrating
that S1 can quantitatively detect cyanide ions. As shown in
Figure 3, S1 coupled with Cu2+ can selectively detect CN− over
other competitive anions. The effect of biological glutathione
(GSH) on the reaction was checked, as shown in Figure S11;
the reaction can proceed in the presence of a low
concentration of GSH, but the conversion yield is inferior to
that in the absence of GSH, suggesting the existing of GSH
interference. It is presumed that the more Cu(I) produced by
GSH would decrease the oxidation capability of the cyanide-
Figure 3. (a) Fluorescence spectra of S1 (1 μM) in CH3CN/PBS
buffer (v/v = 2:1, 10 mM, pH 7.4) mixtures containing 8 equiv of
Cu2+ upon addition of CN− and other anions. (b) Fluorescence
intensity value of S1 upon addition of 50 equiv of various anions
(black bars). Red bars represent the changes after the subsequent
addition of 50 equiv of CN−.
copper complex. The effect can be screened to some extent by
subsequent addition of biological reductant ascorbic acid or
H2O2 that can transfer Cu(I) to Cu(II), despite the fact that
the condition still needed to be optimized.
In order to further study the possibility of the synthesis of
benzazoles from Schiff bases and the detection of the copper-
cyanide complex in a biological environment, we carried out
fluorescence imaging experiments in HepG2 cells. HepG2 cells
were first incubated with compound S1 (1 μM) in the culture
media at 37 °C for 30 min. As shown in Figure 4, the cells
showed no obvious fluorescence signal. The control groups
were further incubated with 20 μM CN− or 20 μM Cu2+ alone
for 30 min. It was found that these cells had no fluorescence
intensity change. In sharp contrast, strong blue fluorescence in
the cells was observed as these cells were treated with the
copper-cyanide complex for 20 min. The results showed that
compound S1 had good cell membrane permeability and could
in situ form fluorescent benzazoles for detection of CN− and
Cu2+ in living cells. It should be noted that this copper-cyanide
catalyzed reaction can proceed even in the presence of
biological GSH probably due to the fact that there is a complex
redox balance in the biological system. We further performed
colocalization experiments using commercial mitochondrial
tracker Rhodamine 123, endoplasmic reticulum ER-tracker
red, and lysotracker green DND-26, respectively (Figure S12).
C
Org. Lett. XXXX, XXX, XXX−XXX