Fluorescent Probes
FULL PAPER
H3PO4 as the external standard. All melting points were measured on a
Yanaco micro-melting point apparatus (Yanagimoto MFG Company),
and are given (uncorrected).
shown in Figure 6d. The cells, which were PMA-stimulated
for 12 h, were further treated with a solution of Tiron
(Tiron, 1,2-dihydroxy-3,5-benzenedisulfonic acid disodium
3’,6’-Bis(diphenylphosphinyl)fluorescein (PF-1): Freshly distilled triethyl-
amine (2.0 mL) was added to a solution of Ph2P(O)Cl (2.37 g, 10 mmol)
in anhydrous THF (25 mL) and then, dropwise with stirring, a solution of
fluorescein (1.66 g, 5 mmol) in anhydrous THF (50 mL). The reaction
mixture was heated for 30 min with mild boiling of the solvent. Stirring
and cooling to room temperature, the precipitated triethylamine hydro-
chloride was separated by filtration. The solvent of the resulting solution
was removed by evaporation, and the residue was dissolved in anhydrous
benzene (50 mL). The solution was washed with 5% ammonia, water and
saturated aqueous NaCl solution. The organic layer was dried over anhy-
drous Na2SO4, and concentrated in vacuo. The residue was dissolved in
benzene, and insoluble solid was filtered at room temperature. Then
hexane was dropped slowly to the filtrate until a precipitate formed,
which was filtered and dried in vacuum. The solid powder obtained was
subjected to silica gel chromatography eluted with ethyl acetate/hexane
3:1 to afford PF-1 (2.56 g, 70%) as a white solid. M.p. 118–1208C;
CÀ
salt, a cell-permeable O2 scavenger[23]) for 1 h and incubat-
ed with PF-1 for 10 min, the fluorescence intensity was
markedly suppressed in Figure 6e; the phase contrast image
is given in Figure 6f. The specificity of the measurement is
confirmed by adding a nonenzymatic superoxide scavenger,
Tiron. Brightfield transmission light measurements in Fig-
ure 6d confirms that the cells are viable throughout the
imaging experiments. These results establish that PF-1 is
membrane-permeable, and can specially respond to the mi-
CÀ
cromolar change of O2 concentrations within living cells,
which demonstrate that the probe is an excellent sensor to
superoxide anion radical.
3
1H NMR (300 MHz, [D6]DMSO/CDCl3 20:1, 258C, TMS): d=8.01 (d, J-
AHCTREUNG
Conclusion
AHCTREUNG
A
ACHTREUNG
In summary, we describe the chemical synthesis, property
analysis and biological application of the diphenylphosphi-
nate fluorescein (PF-1), a new type of phosphinate-based
=
31.384 ppm; 1H NMR and MS spectra were shown in Figures S7 and S8
(see Supporting Information); IR (KBr): n˜ =1768 (lactone, C=O), 1606,
1486 cmÀ1 (phenyl); elemental analysis calcd (%) for C44H30O7P2 (732.2):
C 72.14, H 4.13; found: C 72.26, H 4.15; MS (APCI): m/z: calcd 732.2,
found 732.2.
CÀ
fluorescent probe for imaging O2 in biological environ-
ments. The synthesis method is simple and original. Photo-
metric and fluorimetric experiments of the probe show that
the sensitive fluorogenic reagent features a high selectivity
CÀ
Product analysis of the reaction of PF-1 with O2
: KO2 (71.0 mg,
1.0 mmol) was added to a solution of the probe PF-1 (73.2 mg, 0.1 mmol)
in 10%DMSO-PBS (20 mL). The reaction solution was stirred for 10 min
and cooled to room temperature. Excitation and emission spectra of the
solution diluted with PBS buffer (0.1m, pH 7.4) confirm that fluorescein
CÀ
for O2 over other intracellular ROS and biological com-
pounds, a wide response range and low detection limit
owing to its nucleophilic mechanism. Using fluorescence mi-
CÀ
is the main product generated from the reaction between PF-1 and O2
CÀ
croscope, cell-derived O2 were located in living cells. The
(lex/lem =490/530 nm), while the byproduct, a white acicular solid, can be
obtained by vaporizing automatically the reactive solution. M.p. 191–
1928C; 1H NMR (300 MHz, [D6]DMSO, 258C, TMS): d=7.75–7.72 (m,
8H; CH), 7.52–7.43 (m, 12H; CH), 5.56 ppm (s, 1H; OH); 31P NMR:
34.193 ppm. The solid should be diphenylphosphinic acid.
CÀ
specific response of PF-1 to O2 was confirmed by adding a
nonenzymatic superoxide scavenger. These experimental re-
sults show that PF-1 is an excellent fluorescent probe, which
possesses good selectivity, high sensitivity, good water solu-
bility, and prompt reactivity. Current efforts are devoted
toward applying PF-1 to different biological systems to ex-
plore its potential applications. We believe that such a phos-
phinate-based fluorescent dye will have a great application
in detecting oxidative stress through direct intracellular
imaging. The ability to detect superoxide by intact cells will
facilitate investigations of the generation, metabolism, and
mechanisms of superoxide-mediated cellular homeostasis
and injury.
Spectroscopic materials and methods: Water was purified using a arium
611 VF system with ultrafilter and UV lamp (Sartorius, Germany). Xan-
thine oxidase (XO), xanthine (X), l-glutathione reduced (GSH),
tBuOOH (aq 70%), H2O2 (aq 30%), NaOCl (aq 5%), DMSO, KO2, 3-
morpholinosydnonimine hydrochloride (SIN-1), 3-(aminopropyl)-1-hy-
droxy-3-isopropyl-2-oxo-1-triazene (NOC-5), superoxide dismutase
(SOD), phorbol 12-myristate 13-acetate (PMA), phosphate buffered
saline (PBS), and RPMI 1640 medium were purchased from Sigma
Chemical Co. 1,4-Hydroquinone was purchased from Fluka Chemical Co.
Kunming Balb/c mouse were purchased from the animal experimental
center of the Shandong University. Mice used in this study were 6–12
weeks old. 1,2-Dihydroxy-3,5-benzenedisulfonic acid disodium salt
(Tiron) were purchased from Shanghai sss reagent Co. LTD.
Sample preparation: Probe solutions (0.1 mm) were prepared immediate-
ly before use by diluting a stock solution of PF-1 (1 mm) in DMSO with
pH 7.4, 0.1m PBS buffer (the miscibility of probe solution in organic sol-
vents with aqueous systems was markedly improved compared with that
reported[24]). Solutions of XO (0.5 UmLÀ1 for evaluating specificity and
obtaining calibration curves), xanthine (1.0 mm), SOD (1500 UmLÀ1),
GSH (1.0 mm), H2O2 (1.0 mm), CoSO4 (1.0 mm), NaClO (1.0 mm),
tBuOOH (1.0 mm), SIN-1 (1.0 mm), 4-hydroquinone (HQ, 1.0 mm) were
prepared with H2O. NOC-5 was used as a solution (1.0 mm) in aqueous
10 mm NaOH. KO2 was used as a solution (1.0 mm) in DMSO.
ExperimentalSection
Synthetic materials and methods: Silica gel (100–200 mesh, Qingdao
Haiyang Chemical Co.) was used for column chromatography. Analytical
thin-layer chromatography was performed using GF254 silica gel (pre-
coated sheets, 0.77 mm thick, Taizhou Si-Jia Biochemical Plastic Compa-
ny). Diphenylphosphinyl chloride (Ph2P(O)Cl) and fluorescein were pur-
chased from Sigma Chemical Co. All the other chemicals were purchased
1
from Chinese Chemical Co. H NMR spectra were recorded on a Bruker
Fluorescence analysis: Fluorescence emission spectra were obtained with
FLS-920 Edingburgh Fluorescence Spectrometer with a Xenon lamp and
1.0 cm quartz cells. Into a 10 mL color comparison tube were added PBS
buffer (2.0 mL, 0.1m), PF-1 (1.0 mL, 0.1 mm), and different concentra-
tions of xanthine plus xanthine oxidase or KO2. After diluted to the
Avance 300 MHz. Mass spectra were measured with a Agilent HP 1100
LC-MSD (U.S). The IR spectrum was obtained on a Bruker Tensor-27.
Elemental analysis was performed on Perkin Elmer Series CHNS/O Ana-
lyzer. 31P NMR spectra were measured on a Bruker DPX-400 with a
working frequency of 161.9 MHz using DMSO as the solvent and 85%
Chem. Eur. J. 2007, 13, 1411 – 1416
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1415