G. He et al. / Tetrahedron xxx (2016) 1e6
3
0.04 mol) and piperidine (2 mL) were mixed in 60 mL anhydrous
ethanol. The mixture was refluxed for 6 h, cooled to room tem-
perature, and evaporated to give compound 3 as orange oil, which
was used directly for the next step.
dissolved completely in CH3CN:HEPES (3:2, v/v) buffer, as shown in
Fig. S12. Therefore, this ratio is suitable for the spectral
experiments.
3.2. The dependence of pH
2.3.2. Synthesis of 7-(diethylamino)-2-oxo-2H-chromene-3-
carbohydrazide14b
The effect of pH on the fluorescence intensity for compound 1
m
M) treated with Cu2þ (4 equiv.) and GSH (2 equiv.) in
Compound 3 (4.3399 g, 15 mmol) and hydrazine monohydrate
(3.64 mL, 60 mmol, 80%) were added into an ethanol (40 mL) so-
lution. The mixture was stirred for 12 min at room temperature.
After cooling in an ice bath for 15 min, the precipitate was filtered
and purified by chromatography (mobile phase, ethyl acetate) to
give compound 2 as orange needles, mp 161e162 ꢂC. Yield: 83%.
(10
CH3CN:HEPES (3:2, v/v) buffer was showed in Fig. S13. The fluo-
rescence intensity changed less than 10% in the range of 6.0e8.0,
which indicated that compound 1 could be applied in physiological
condition.
3.3. The UVevis absorption and fluorescence spectra responses of
compound 1 to Cu2þ ions
2.3.3. Synthesis of compound 1
Compound 2 (0.3028 g, 1.1 mmol) and 2,6-pyridine dicarbox-
aldehyde (0.0676 g, 0.5 mmol) were dissolved in ethanol (30 mL).
After refluxed for 12 h under nitrogen and cooled to room tem-
perature, the obtained yellow precipitate was filtered, washed and
The UVevis absorption spectrum of compound 1 (10
mM) in
CH3CN:HEPES (3:2, v/v) solution was shown in Fig.1. The maximum
UVevis absorption peak of compound 1 was at 440 nm. Upon
addition of Cu(ClO4)2, the UVevis absorption peak of compound
1 at 440 nm gradually decreased. After the addition of 6 equivalents
of Cu2þ, its peak value reached equilibrium. The UVevis absorption
dried to afford compound 1. 1H NMR (400 MHz, CF3COOD):
d (ppm)
9.53 (s, 2H), 8.95 (s, 2H), 8.92e8.79 (m, 1H), 8.73e8.56 (m, 2H),
8.44e8.31 (m, 2H), 8.04e7.94 (m, 2H), 7.86e7.81 (m, 2H), 3.99 (q,
J ¼ 6.9 Hz, 8H), 1.40 (t, J ¼ 7.0 Hz, 12H); 13C NMR (100 MHz,
spectra of compound 1 (10 m
M) with other metal ions (such as Naþ,
CF3COOD):
d (ppm) 155.1, 152.2, 148.7, 145.9, 142.2, 140.4, 139.9,
Mg2þ, Kþ, Ca2þ, Cr3þ, Mn2þ, Fe3þ, Fe2þ, Co2þ, Ni2þ, Cu2þ, Cuþ, Zn2þ
,
133.8, 127.9, 120.3, 119.7, 117.5, 115.2, 111.7, 55.5, 9.0. ESI-MS: m/z:
Agþ, Cd2þ, Hg2þ, Pb2þ, as ClO4ꢁ salt) were shown in Fig. S14. The
result indicated that Cu2þ showed the most significant response to
compound 1 in the UVevis absorption spectrum, other metal ions
had weak impacts on the absorption.
650.2672, [compound 1 þ H]þ; 672.2513, [compound 1 þ Na]þ.
2.3.4. Synthesis of probe compound 1-Cu2þ and other complexes
Compound 1 (0.0065 g, 0.01 mmol) and excess Cu(ClO4)2$6H2O
(0.0074 g, 0.02 mmol) were dissolved in ethanol (20 mL). After
refluxed for 30 min, the obtained precipitate was filtered, washed
and dried to afford compound 1-Cu2þ. Yield: 79%. ESI-MS: m/z:
711.1933. In order to investigate the behaviors of compound 1 with
other metal ions, complexes such as compound 1-Co2þ, compound
1-Zn2þ, and compound 1-Mg2þ were also prepared in a similar
method, and further characterized by ESI-MS (Figs. S8eS10).
The fluorometric titration experiment of compound 1 (10 mM)
with Cu2þ was proceeded in CH3CN:HEPES (3:2, v/v) solution
(Fig. 2). compound 1 exhibited a strong fluorescence emission peak
at 485 nm (excited at 440 nm). Upon addition of Cu(ClO4)2, the
fluorescence intensity of compound 1 gradually decreased. After
the addition of 4 equivalents of Cu2þ, the peak intensity reached
equilibrium. The low fluorescence intensity of the compound 1-
Cu2þ might be due to the quenching effect resulted from the
paramagnetism and photoinduced electron transfer of the copper
ion.15 As a comparison, the fluorescence responses of compound 1
3. Results and discussions
to the other metal ions (such as Naþ, Mg2þ, Kþ, Ca2þ, Cr3þ, Mn2þ
,
Fe3þ, Fe2þ, Co2þ, Ni2þ, Cu2þ, Cuþ, Zn2þ, Agþ, Cd2þ, Hg2þ, Pb2þ, as
ClOꢁ4 salt.) were also investigated (Fig. S15), the results were
3.1. The experiment of water solubility
Compound 1 has a relatively high fluorescence intensity and
Fig. 1. The UVevis spectra titration of compound 1 (10
m
M) upon addition of Cu2þ in
Fig. 2. The fluorescence spectra titration of compound 1 (10 mM) upon addition of
CH3CN:HEPES (3:2, v/v) solution. Inset: UVevis titration profile of compounds 1 upon
Cu2þ in CH3CN:HEPES (3:2, v/v) solution. Insert: fluorescence titration profile at
addition of Cu2þ, the absorption spectra were recorded at 440 nm.
485 nm upon the addition of Cu2þ (excited at 440 nm).
Please cite this article in press as: He G, et al., Synthesis of a fluorogenic probe for thiols based on a coumarin schiff base copper complex and its