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yellow uorescence can be observed under excitation at 557 nm. MHz, DMSO, 25 ꢀC): d ¼ 7.75 (s, 1H), 7.47 (s, 2H), 6.98 (s, 1H),
Furthermore, the color changes from yellow to deep pink 6.33 (d, 6H, J ¼ 9.0 Hz), 3.31 (d, 8H, J ¼ 6.0 Hz), 2.94 (d, 2H, J ¼
quickly and clearly, aer adding Fe3+ in P1. In the uorescence 9.0 Hz), 2.18 (d, 2H, J ¼ 6.0 Hz), 1.07 (t, 12H, J ¼ 6.0 Hz) ESI-MS:
titration experiments, the uorescence intensity remained m/z ¼ 485.20 [M + H]+.
unchanged even aer the concentration of the Fe3+ was more
Synthesis of compound P1. Into compound b (1.21 g, 2.5
than 1 equiv. Moreover, the biological experiment proves that mmol) in ethanol solution (25 mL) in a 100 mL round-bottomed
P1 could perform in living cells, showing its potential for use in ask was added compound a (0.43 g, 2.5 mmol). The solution
practical and a wide range of applications.
was then heated at reux overnight. Aer cooling to room
temperature, a yellow solid precipitated out. The solid was
washed by cool ethanol until the ltrate turned colorless, then
2. Experimental
2.1. Materials and apparatus
1
dried in vacuo. Yield: 1.12 g (70%), yellow powder P1. H-NMR
(300 MHz, DMSO, 25 ꢀC): d ¼ 13.84 (s, 1H), 8.75 (d, 1H, J ¼
8.5 Hz), 7.38–7.90 (m, 6H), 7.16 (s, 1H), 7.01 (s, 1H), 6.69 (d, 2H,
J ¼ 9.2 Hz), 6.29 (dd, 5H, J ¼ 6.0, 6.0 Hz), 3.42 (s, 4H), 3.22
(d, 8H, J ¼ 5.7 Hz), 1.02 (s, 12H) ppm. 13C-NMR (75 MHz, DMSO,
25 ꢀC): d ¼ 176.42, 167.99, 159.71, 153.94, 153.08, 148.83,
137.21, 134.49, 133.29, 130.57, 129.28, 128.78, 128.65, 128.21,
125.80, 125.42, 124.09, 122.90, 122.64, 118.81, 108.62, 106.59,
105.19, 97.70, 64.64, 49.70, 44.08, 40.99, 12.79 ppm. ESI-MS:
m/z ¼ 639.30 [M + H]+ 661.30 [M + Na]+ 677.25 [M + K]+.
All of the chemicals were from commercial suppliers. The
solvents (methanol, ethanol) were dried and distilled before
use. All of the other chemicals were of analytical reagent grade,
unless otherwise stated. As for the aqueous solutions, the water
was puried by a Milli-Q Advantage Water Purication System.
1H NMR and 13C-NMR spectra were recorded using a Bruker
Ultrashield 300 MHz spectrometer operating at 300 and 75
MHz, respectively. Samples were in deuterated dimethyl
sulphoxide (DMSO-d6) as the solvent. Chemical shis were
expressed in ppm, and TMS was the internal standard. Mass
spectroscopy was carried out by Micromass GC-TOF and Agilent
Technologies 6540 UHD Accurate-Mass Q-TOF LC/MS instru-
ments. An Hitachi Fluorescence spectrophotometer-F-4600 was
used to measure the uorescence spectra. A Varian Cary 50
Probe UV/Vis spectrophotometer was used to measure UV/Vis
spectra. An Olympus FV-1000 laser scanning confocal uores-
cence microscope was used to capture the uorescent images.
2.3. Measurement procedures
Stock solutions of 1.0 ꢁ 10ꢂ3 M P1 and 1.0 ꢁ 10ꢂ3 M Fe3+ were
prepared by dissolving P1 in ethanol–H2O (1 : 1) solution and
Fe2(SO4)3$9H2O in double-distilled water, respectively. The Fe3+
solution was diluted stepwise to yield working solutions with
concentrations ranging from 0.1 mM to 5 mM. The pH range
solutions were prepared by adjusting 50 mM Tris–HCl solution
with HCl or NaOH solution. Solutions of the other metal ions
were prepared by dissolving Al(NO3)3$H2O, LiClO4, NaCl, KCl,
MgCl2, CaCl2, Fe(NO3)2$6H2O, FeCl3$6H2O, CoCl2$6H2O,
Ni(NO3)2$6H2O, AgBF4, Cr(NO3)3$9H2O, Cd(NO3)2$4H2O, ZnCl2,
Hg(ClO4)2$3H2O, CuCl2$2H2O, Pb(NO3)2, Mn(NO3)2, GaCl3,
FeSO4$7H2O, and HgCl2 in double-distilled water (1.0 ꢁ 10ꢂ3 mM).
2.2. Intermediates and monomer synthesis
The overall synthetic steps for the monomer are shown in
Scheme 1.
Synthesis of compound a. Compound a was synthesized
according to an earlier reported article.26
2.4. Cell imaging
Synthesis of compound b. Into rhodamine B (4.79 g, 10
mmol) in ethanol solution (30 mL) in a 100 mL round-bottomed A fresh stock of HeLa cells was seeded into a glass bottom dish
ask was added ethylenediamine (10 mL). The solution was with the density of 1 ꢁ 10ꢂ5 cells per dish, and incubated for 24
heated at reux and stirred for 4 h. Aer cooling to room h. Then, 10 mM compound P1 (198 mL DMEM mixed with 2 mL
temperature, a red solid precipitated resulted and was washed of 1.0 ꢁ 10ꢂ3 M P1 solution in ethanol–H2O) solution was
by cool ethanol until the ltrate turned colorless.1H-NMR (300 added to the cells, and then they were incubated for 15 min at
room temperature. Then the solution was removed and the cells
were washed by PBS (2 mL, three times) to clear any remaining
P1 molecules that did not get inside the cells. Aerwards, 10 mM
Fe3+ DMEM solution was added to the cells, and then the cells
were incubated for 15 min at room temperature. The solution
was removed and the cells were washed by PBS (2 mL, three
times). Fluorescence imaging was performed with a confocal
laser scanning microscopy (lex ¼ 546 nm, uorescent signals
were collected at 550–650 nm). The images were captured by a
photomultiplier.
2.5. Fluorescent imaging of Fe3+ in zebrash
Zebrash were raised at 28 ꢀC and maintained at optimal
breeding conditions. For mating, male and female zebrash
Scheme 1 Schematic of P1 synthesis.
were maintained in one tank at 28 ꢀC on a 12 h light/12 h dark
This journal is © The Royal Society of Chemistry 2014
RSC Adv., 2014, 4, 39984–39990 | 39985