Chemical Papers
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13
some limitations considering the practical applications in
biological systems, such as undesired spectra overlap, long
response time and relatively poor selectivity. Consequently,
H NMR and C-NMR spectra were recorded on a
Bruker AVIII-400 MHz spectrometer. High-resolution
mass spectra (HRMS) were measured with Thermo Scien-
tiꢂc Orbitrap Elite. Absorption spectra were measured using
a Thermo Scientiꢂc BioMate 3S UV/Vis spectrophotom-
eter. Fluorescence measurements were carried out with the
F97pro ꢁuorospectrophotometer.
ꢁ
uorescence probes ꢀor more efective detection oꢀ Cys
should be given additional research attention.
Efective identiꢂcation groups are the key roles ꢀor distin-
guishing Cys over Hcy and GSH. Yang et al. (2011) reported
that acrylates can selectively identiꢀy Cys by cyclization
reaction. Moreover, chloropropionate/bromopropionate ester
and crotonates are the other efective groups ꢀor selective
detection oꢀ Cys (Kim et al. 2014; Murale et al. 2014a,
b). Thus, these methods provided an efective strategy ꢀor
selectively detecting Cys. Coumarin as a natural product is
an excellent ꢁuorescent dye. When the electron-donating
substituent at the 7-position, coumarins can show strong
Results and discussion
Compound 2 was synthesized using the classic Knoev-
enagel reaction with 2,4-dihydroxybenzaldehyde (1) and
ethyl 2-cyanoacetate under the condition oꢀ microwave irra-
diation according to the literature (Valizadeh et al. 2005).
Subsequently, probe 1 was synthesized by compound 2 and
acryloyl chloride in 60% yield (Scheme 1, ꢀor details and
characterization see Supporting Inꢀormation), and conꢂrmed
by NMR and HRMS (ESI).
ꢁ
uorescence. In addition, attaching electron-withdrawing
groups to the 3-position oꢀ coumarin afords greater spectral
redshiꢀts and higher molar extinction coeꢃcients (Liu et al.
2
012). Dai et al. (2014) reported that a ꢁorescence probe
with carbonyl substitution at the 3-position oꢀ coumarin can
selectively detect Cys. However, the reaction time is too long
to track the real-time change oꢀ Cys. Thus, in our work, we
introduced the more electron-withdrawing cyano group at
the 3-position oꢀ coumarin to increase the level oꢀ electron
transꢀer in the molecule and to improve reaction rate/spec-
tral properties. Thereꢀore, we designed and synthesized the
Firstly, the UV–Vis absorption oꢀ probe 1 (10 μM) was
investigated in an aqueous PBS bufer (pH 7.4) (contain-
ing 50% CH CN, v/v). Probe 1 has obvious absorption
3
at 300 and 340 nm, respectively. Upon the addition oꢀ
Cys (100 μM), the absorption bands at 300 and 340 were
decreased with gradual enhancement oꢀ a new absorption
band at 413 nm over time (Fig. S1). Thus, 413 nm was deter-
mined as the ꢁuorescence excitation wavelength. Subse-
quently, we investigated the ꢁuorescence intensity response
oꢀ probe 1 to the diferent amino acids (l-Cys, Hcy, GSH,
Arg, Asp, Tyr, Pro, Lys, Gly, Ala, Phe, Thr, dl-Met, His,
Ser, Leu, Trp, Ile, l-Met, Glu, 100 μM) with an excitation
at 413 nm and 5 min incubation time (Fig. 1a). The result
showed that the ꢁuorescence intensity oꢀ probe 1 was signiꢂ-
cantly increased at 450 nm with the addition oꢀ Cys. How-
ever, other amino acids exhibited negligible change, though
only Hcy and GSH showed slight interꢀerence. Moreover,
to ꢀurther explore the selectivity, we research the reaction
situation oꢀ the probe and millimole level oꢀ GSH (Fig. S2).
Results showed that the ꢁuorescence intensity oꢀ probe 1
was increased with 5 mM GSH, but the intensity was still
lower than that oꢀ Cys (100 μM). The ꢁuorescence intensity
was not ꢀurther increased with the addition oꢀ 6 mM GSH.
Thereꢀore, probe 1 is able to sense selectively Cys under this
test condition.
3
-cyano-2-oxo-2H-chromen-7-yl acrylate (probe 1), which
includes the biothiol reaction site oꢀ acrylate and coumarin
uorophore. Probe 1 can selectively detect Cys over other
ꢁ
thiols within 5 min, and possesses low detection limit and
excellent detection relationship with Cys. Thus, with the
substitution oꢀ the cyano group ꢀor carbonyl substitution at
the 3-position oꢀ coumarin, the reaction rate was dramati-
cally increased and ꢁuorescence intensity change was obvi-
ous. Meanwhile, the results showed that probe 1 was also
capable oꢀ detecting Cys in living cells.
Experimental section
2
,4-Dihydroxybenzaldehyde, ethyl 2-cyanoacetate, acryloyl
chloride and N-ethylmaleimide,3-(4,5-dimethylthiazol-2-yl)-
,5-diphenyltetrazolium bromide (MTT) were purchased
2
ꢀ
rom Sigma-Aldrich. Unless otherwise noted, materials were
purchased ꢀrom commercial suppliers and used without ꢀur-
ther puriꢂcation. All the solvents were puriꢂed and dried
according to general methods.
The ꢁuorescence intensity change oꢀ probe 1 in the pres-
ence oꢀ diferent concentrations oꢀ Cys was explored with an
excitation at 413 nm. Remarkably, with the increase oꢀ Cys
Scheme 1 Synthesis oꢀ probe 1
1
3