employed. Further, 4-phenoxyphenol (4-PPOH) was also used in
this study to mimic the behavior of the reactive center in HPF.
Electron paramagnetic resonance (EPR) experiments
The interaction between HPF and KFC was studied in alkaline
aqueous solution (NaOH 0.1 M). For that purpose, HPF (2 ×
10−4 M) was added to a strongly oxidizing KFC solution (10−1 M),
under air. The reactants were mixed at room temperature directly
in an EPR tube and immediately after, the mixture was frozen in
liquid nitrogen. The measurement was monitored at −160 ◦C.
The hydrogen abstraction from 4-PPOH (80 mM) was carried
out in a mixture of acetonitrile–di-tert-butyl peroxide (1 : 1, v : v).
The sample was deaerated with argon for 15 min then transferred
to an EPR flat cell (JEOL, ES LC 12, 0.3 mm thick) under
an inert atmosphere. The cell was then placed into the cavity
of the spectrometer and irradiated in situ by the means of a
xenon illuminator (Luzchem). A cut-off filter (k > 345 nm) was
placed between the lamp and the sample in order to avoid the
direct excitation of 4-PPOH. The EPR signal was recorded at
room temperature. All measurements were carried out on a JEOL
(FA 100) EPR spectrometer. The different parameters used are
reported in the legends of the different figures. The g-values were
estimated by comparison with the 3rd and the 4th lines of the Mn2+
internal marker, calibrated with the standard DPPH radical (g =
2.0036).
Experimental
Materials
2-[6-(4ꢀ-Hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic
acid
(3ꢀꢀ-(p-hydroxyphenyl)fluorescein, HPF) was purchased from
Molecular Probes (Canada). 4-Phenoxyphenol (4-PPOH), 2,2ꢀ-
azobis-(2-methylpropionitrile) (AIBN), 2,2ꢀ-azobis-(2-methyl-
propionamidine) dihydrochloride (AAPH), sodium hydroxide
(NaOH), di-tert-butyl-peroxide were purchased from Aldrich
(Canada). Potassium ferricyanide (KFC) was supplied by Sigma
(Canada). Acetonitrile from Omnisolv was HPLC grade.
Fluorescence and absorption measurements
Fluorescence emission spectra were recorded using an LS-50
fluorimeter (Perkin Elmer). The excitation of the HPF probe or its
products was set at 490 nm. When necessary, the temperature was
maintained at 40 ◦C using a cuvette holder equipped for circulating
thermostated water.
Absorption spectra were recorded using a Cary 50 (Varian)
single beam spectrophotometer.
Results
For both measurements, the solutions were placed in a 1 ×
1 cm quartz cuvette. Prior to the experiments, the samples were
deaerated by bubbling argon through the solution. The oxygenated
samples were obtained by maintaining an oxygen atmosphere
above the solution. All gases were supplied by Proxair (Canada)
with a high purity.
Although HPF was described as non-reactive towards the peroxyl
radical in the literature,8 we demonstrated the ability of the probe
to react with this type of ROS. Indeed, as depicted in Fig. 1, an
increase in the fluorescence is observed when HPF (0.8 lM) is
in presence of AAPH (10 mM) at 40 ◦C under O2. AAPH is a
convenient water-soluble source of carbon-centered radicals that
react with oxygen readily to yield peroxyl radicals.9,10
Laser flash photolysis
The phenoxyl radical form (4-PPO•) from 4-PPOH was generated
by laser excitation of di-tert-butyl-peroxide in acetonitrile (50 :
50) using 355 nm pulses from a Continuum Nd-YAG Surelite.
The instrument is a customized version of a Luzchem LFP-111
system (Luzchem Research, Ottawa, Canada). All experiments
were carried out using static Luzchem cuvettes constructed from
7 × 7 mm fused silica tubing. Samples were deaerated with argon
prior to exposure.
Chromatographic analysis
The characterization of the by-product resulting from the inter-
action of 4-PPOH and the peroxyl radical was performed by
HPLC (Waters, model 2690) with UV–vis (Waters, model 996) and
mass spectroscopy detection (Waters, Integrity system). Briefly, a
solution of 4-PPOH (10−3 M) dissolved in acetonitrile in presence
of AIBN (10−1 M) was kept under oxygen at 40 ◦C for 3 h in a water
bath. The mixture was then injected without any treatment on a C-
18 reverse phase column (Zorbax, SB-C18 4.6 × 250 mm, Agilent).
An isocratic elution with a mixture of water and acetonitrile (50 :
50) was realized at a flow rate of 0.25 ml min−1 for 40 min, followed
by a gradient to reach 100% acetonitrile in 20 min. Notably,
p-benzoquinone (the expected product if 4-PPO• underwent a
cleavage analogous to that of Scheme 1), was not detected. All
the solvents used as mobile phase were of HPLC grade.
Fig. 1 Bottom: evolution of the maximum fluorescence at 511 nm of
a HPF (0.8 lM) solution in phosphate buffer (pH 7.4) in the presence
of AAPH (10 mM) under O2 at 40 ◦C. Top: absorption (thick, A), the
excitation (E) and the fluorescence (F, right axis) of a HPF (1.67 lM)
solution in phosphate buffer (pH 7.4).
This journal is
The Royal Society of Chemistry 2006
Org. Biomol. Chem., 2006, 4, 802–807 | 803
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