X. Bao et al. / Bioorg. Med. Chem. 22 (2014) 4826–4835
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applications. Herein, we report the synthesis and characterization
of a novel Rhodamine B-derivatized fluorescent chemosensor
based on a pyrrole moiety, named RBPY (Fig. 1). Our findings show
that RBPY exhibits a high selectivity and sensitivity toward Fe3+
ions in a MeOH/H2O (3:2, v/v, pH 7.10, HEPES buffer, 0.1 mM) solu-
tion. The formation of RBPY–Fe3+ complexes is fully reversible in
the presence of sulfide anions and could also be used as an efficient
sensor for S2ꢀ under the same conditions. Moreover, the fluores-
cence microscopy experiments in this study also demonstrated
that RBPY can be used as a fluorescent probe for the detection of
Fe3+ in living cells.
mixture was refluxed for 12 h and then taken to dryness under vac-
uum. The residue was dissolved in CH2Cl2 and then washed with
H2O and brine. The organic layer was dried with MgSO4. After
the removal of the solvent, flash chromatography (silica gel;
MeOH/CH2Cl2, 3:97; v/v) of the residue yielded 1 as a pink solid
(3.2 g, 83%). 1H NMR(CDCl3), d 8.12–7.78 (m, 1H), 7.64–7.34 (m,
2H), 7.19–6.96 (m, 1H), 6.43 (d, 2H, J = 8.9 Hz), 6.37 (d, 2H,
J = 2.5 Hz), 6.27(dd, 2H, J1 = 2.6, J2 = 2.5 Hz), 3.66–3.24 (m, 8H),
3.28–2.91(m, 2H), 2.41 (t, 2H, J1 = 6.7 Hz, J2 = 6.6 Hz), 1.40 (s, 2H),
1.16 (t, 12H, J1 = 7.0 Hz, J2 = 7.1 Hz) ppm. 13C NMR (CDCl3), d
168.59, 153.43, 153.26, 148.79, 132.37, 131.20,128.64, 128.01,
123.79, 122.70, 108.13, 105.64, 97.71, 77.38, 77.13, 76.88, 64.91,
44.31, 43.80, 40.73, 12.56 ppm. ESI–MS (M++1) found, 485.13;
calcd for C30H37N4O+2, 485.29.
2. Experimental
2.1. Materials and general methods
2.3. Synthesis of 2-(2-(((1H-pyrrol-2-yl)methyl)amino)ethyl)-
30,60-bis(diethylamino)spiro[isoindoline-1,90-xanthen]-3-one
(RBPY)
All reagents and organic solvents used were of ACS grade or
higher and were used without further purification. Unless other-
wise noted, all chemicals were purchased from J&K Scientific
(Shanghai, China) and were used as received. All solvents were of
analytical grade, and double-distilled water was used in all
experiments. The salts used to prepare metal ion stock solutions
were AgNO3, AlCl3, Ba(NO3)2, CaCl2, CdCl2ꢁ2.5H2O, CoCl2ꢁ6H2O,
CrCl3ꢁ6H2O, IrCl3, CuCl, CuCl2ꢁ2H2O, FeCl2ꢁ4H2O, FeCl3ꢁ6H2O, HgCl2,
KCl, LiClꢁH2O, MgCl2ꢁ6H2O, MnCl2ꢁ4H2O, NaCl, NH4Cl, NiCl2ꢁ6H2O,
PbCl2, SnCl2ꢁH2O and ZnCl2. The reactions were performed
under an argon atmosphere using standard Schlenk techniques.
Thin-layer chromatography was performed on a HAIYANG silica
gel F254 plate, and compounds were visualized under UV light
(k = 254 nm). Column chromatography was performed using
HAIYANG silica gel (type: 200–300 mesh ZCX-2).
To a solution of compound 1 (200 mg, 0.413 mmol) in 5 mL of
methanol was added 1H-pyrrole-2-carbaldehyde (117.7 mg,
1.24 mmol). The reacting mixture was stirred at room temperature
for 12 h, followed by the addition of sodium triacetoxyborohydride
(175 mg, 0.826 mmol), and stirred for an additional 3 h. The reac-
tion mixture was then taken to dryness under vacuum. The residue
was dissolved in CH2Cl2 and then washed with H2O. The organic
layer was dried with MgSO4 and then concentrated. Flash chroma-
tography (silica gel; MeOH/CH2Cl2, 3:97; v:v) of the crude mixture
afforded RBPY (181.3 mg) in 78% yield as a yellow solid. 1H NMR
(CDCl3), d 9.36 (s, 1H), 7.90 (dd, J1 = 4.15 Hz, J2 = 3.25 Hz, 1H),
7.62–7.34 (m, 2H), 7.24–7.03 (m, 1H), 6.73 (s, 1H), 6.39 (dd,
J1 = 8.85 Hz, J2 = 2.35 Hz, 4H), 6.25(dd, J1 = 2.35 Hz, J2 = 2.4 Hz,
2H), 6.05 (dd, J1 = 2.7 Hz, J2 = 2.7 Hz, 1H), 5.92(s, 1H), 3.67 (s, 2H),
3.41–3.24 (m, 10H), 2.37 (t, J1 = 6 Hz, J2 = 6.15 Hz, 2H), 1.16 (t,
J1 = 7 Hz, J2 = 7.05 Hz, 12H) ppm. 13C NMR (CDCl3), d 168.80,
153.53, 153.31, 148.82, 133.08, 132.45,131.18, 130.74, 130.00,
128.72, 128.47, 128.07, 123.83, 122.73, 119.24, 117.23,110.90,
108.51, 108.14, 107.89, 107.55, 105.88, 105.53, 103.78, 97.74,
77.30,77.05, 76.80, 66.30, 65.08, 53.34, 51.16, 46.88,45.08, 39.37,
38.24, 12.59 ppm. HRMS (M++1) found, 564.3351; calcd for
C35H42N5O+2, 564.3333.
1H (500 MHz) and 13C NMR (126 MHz) spectra were recorded
on an Avance 500 spectrometer (Bruker; Billerica, MA, USA). The
chemical shifts are reported in d units (ppm) downfield relative
to the chemical shift of tetramethylsilane. The abbreviations br, s,
d, t and m denote broad, singlet, doublet, triplet and multiplet,
respectively. Mass spectra were obtained with a Finnigan TSQ
Quantum LC/MS Spectrometer. High-resolution mass spectra
(HRMS) were acquired under electron ionization conditions with
a double-focusing high-resolution instrument (Autospec; Micro-
mass Inc.). The pH levels of stock solutions were measured using
a PHS-25C Precision pH/mV Meter (Aolilong, Hangzhou, China).
UV–vis and fluorescence spectra were obtained on a UV-3600
UV–vis-NIR spectrophotometer (Shimadzu, Japan) and an Edin-
burgh FLS920 fluorescence spectrophotometer (Livingston, UK),
respectively, at room temperature.
2.4. Stock solution preparation for spectral detection
Stock solutions (10ꢀ3 M) of the chloride or nitrate salts of Ag+,
Al3+, Ba2+, Ca2+,Cd2+, Co2+, Cr3+, Ir3+, Cu+, Cu2+, Fe2+, Fe3+, Hg2+, K+,
Li+, Mg2+, Mn2+, Na+, NH4+, Ni2+, Pb2+, Sn2+, and Zn2+ in MeOH/H2O
(3:2, v/v, pH 7.10) were prepared. The stock solution of RBPY
(10ꢀ3 M) was prepared in MeOH/H2O (3:2, v/v, pH 7.10). Working
solutions of RBPY were freshly prepared by diluting the highly
concentrated stock solution to the desired concentration prior to
spectroscopic measurements.
2.2. Synthesis of 2-(2-aminoethyl)-30,60-bis(diethylamino)spiro-
[isoindoline-1,90-xant hen]-3-one (1)
To a solution of Rhodamine B (3.8 g, 7.95 mmol) in 100 mL of
ethanol was added ethane-1, 2-diamine (5 mL, 10 equiv). The
2.5. UV–vis and fluorescence spectral studies
All experiments were carried out in a CH3OH/H2O solution (3:2,
v/v, pH 7.10, HEPES buffer, 0.1 mM). In each titration experiment, a
10 lM solution of the probe RBPY was placed in a quartz optical
cell with a 1-cm optical path length, and the appropriate amount
of the ion stock solution was added to the quartz optical cell using
a micropipette. Spectral data were recorded 10 min after the addi-
tion of the ions. In the selectivity experiments, the test samples
were prepared by placing an appropriate amount of the cation
stock solution in 3 mL of the probe RBPY solution (10 lM). In the
fluorescence measurements, excitation was provided at 558 nm
Figure 1. Chemical structure of RBPY.
and emission was collected from 565 to 680 nm.