Novel glutathione-linked nitrones as dual free radical probes

Article abstract of DOI:10.1039/c1nj20033j

The detection and identification of transient radicals in biological systems is of importance for the understanding of their roles in a variety of biological processes. Electron paramagnetic resonance spectroscopy coupled with the use of the spin trapping technique has been an indispensable tool for this application owing to its high specificity. In this study, we developed a general method using dual function free radical probes (GS-PBN and its phosphorylated analogue GS-PPN) for the simultaneous determination of transient radicals and the microenvironment where the corresponding spin adducts are situated. This conception was initially proved by high spectral sensitivity of the p-ClPh ^? spin adduct of GS-PBN towards its rotational motion in the glycerol-water system. Results showed that a relatively bulky glutathionyl group in the spin adduct plays an important role in its high sensitivity to the molecular motion. This was further verified by high sensitivity of the p-ClPh^? spin adduct of the newly synthesized probe GS-PPN to its molecular motion. Unlike GS-PBN, GS-PPN can be used to detect O ₂^?- generated in the enzymatic system and PSII membranes of chloroplasts. Based on the relationship between the τ_C values of the superoxide spin adduct and the medium viscosity, the local environment of the adduct in the PSII membranes was determined to be similar to that of the aqueous solution containing ～15% glycerol (η ≈ 1.33 p). The Royal Society of Chemistry and the Centre National de la Recherche Scientifique.

Full text of DOI:10.1039/c1nj20033j

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PAPER
Novel glutathione-linked nitrones as dual free radical probesw
ab
ab
Yangping Liu,z Yuguang Song,z Libo Du,a Frederick A. Villamena,b
Yiqiong Ji,a Qiu Tian,a Ke-Jian Liu*c and Yang Liu*a
Received (in Montpellier, France) 18th January 2011, Accepted 29th March 2011
DOI: 10.1039/c1nj20033j
The detection and identification of transient radicals in biological systems is of importance for the
understanding of their roles in a variety of biological processes. Electron paramagnetic resonance
spectroscopy coupled with the use of the spin trapping technique has been an indispensable tool
for this application owing to its high specificity. In this study, we developed a general method
using dual function free radical probes (GS-PBN and its phosphorylated analogue GS-PPN) for
the simultaneous determination of transient radicals and the microenvironment where the
corresponding spin adducts are situated. This conception was initially proved by high spectral
sensitivity of the p-ClPhꢀ spin adduct of GS-PBN towards its rotational motion in the
glycerol–water system. Results showed that a relatively bulky glutathionyl group in the spin
adduct plays an important role in its high sensitivity to the molecular motion. This was further
verified by high sensitivity of the p-ClPhꢀ spin adduct of the newly synthesꢁized probe GS-PPN to
ꢀ
its molecular motion. Unlike GS-PBN, GS-PPN can be used to detect O2 generated in the
enzymatic system and PSII membranes of chloroplasts. Based on the relationship between the tC
values of the superoxide spin adduct and the medium viscosity, the local environment of the
adduct in the PSII membranes was determined to be similar to that of the aqueous solution
containing B15% glycerol (Z E 1.33 p).
they are and where they are produced, is critical for the
Introduction
understanding of the biological processes in which they are
implicated.
Free radicals including a superoxide anion radical (O2 ꢁ) and
nitric oxide have aroused much interest as toxic species and
also as signaling molecules in pathological and physiological
processes.1–5 Superoxide is often proposed as the primary
radical of a free radical chain in the biomembrane. The
single-electron reduction of molecuꢁlar oxygen by semiquinones
ꢀ
Electron paramagnetic resonance (EPR) spectroscopy
coupled with the use of the spin trapping technique has been
an indispensable tool for the detection of transient radicals
owing to its high specificity.15 In general, a nitrone spin trap
reacts with a reactive radical resulting in a relatively more
persistent nitroxide adduct that can be easily detected by EPR.
ꢀ
results in the formation of O2
in mitochondria6–8 and
chloroplasts,9–12 and this process has been considered as one
In the past decades, main effort ꢁhas been exerted to develop
ꢁ
of the most important O2 sources in cells.13,14 Thus, an
ꢀ
ꢀ
spin traps with improved O2
reactivity and enhanced
ꢁ
ꢀ
accurate and reliable technique for the detection of O2 and
its derived radicals, including the identification of what
stability of the corresponding spin adducts by modifying the
molecular skeleton of the nitrones16–20 or via covalent or
noncovalent binding of the nitrones to cyclodextrin21–25 and
calix[4]pyrrole.26 In addition, much interest has also been
involved in the synthesis of spin traps that may selectively
accumulate into subcellular compartments such as
mitochondria27–29 and cell membranes,30 as well as target
lectins31,32 and cysteine-containing polypeptides.33
a State Key Laboratory for Structural Chemistry of Unstable and
Stable Species, Center for Molecular Science, Institute of Chemistry,
Chinese Academy of Sciences, Zhongguancun, Beijing 100190,
P. R. China. E-mail: yliu@iccas.ac.cn
b Center for Biomedical EPR Spectroscopy and Imaging,
The Davis Heart and Lung Research Institute, The Division of
Cardiovascular Medicine, Department of Internal Medicine, and
Department of Pharmacology, College of Medicine, The Ohio State
University, Columbus, OH 43210, USA
Since the nitroxide adduct from reaction of a spin trap with
free radicals has a very similar structure with the commonly
used nitroxide spin label, it can be expected that the EPR
spectral profile of the nitroxide adduct is sensitive to its
molecular motion.34 Therefore, the nitroxide adduct could
not only be used to determine the nature of the trapped
radical, but also potentially serve as a ‘‘reporter molecule’’
c College of Pharmacy, University of New Mexico, 2502 Marble NE,
Albuquerque, NM 87131, USA. E-mail: kliu@salud.unm.edu
w Electronic supplementary information (ESI) available: Kinetics of
decay of GS-PPN–OOH and EPR hyperfine splitting constants of
GS-PPN spin adducts. See DOI: 10.1039/c1nj20033j
z Both authors contributed equally to this work.
c
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Chart 1 Molecular structure of spin traps and the spin label
TEMPOL.
for the system under investigation. However, to the best of our
knowledge, the potential application of the spin adduct in
quantitatively determining the local environment has never
been reported, although there are few examples regarding
protein spin labeling by trapping protein radicals with spin
traps.35,36
With this in mind, we herein investigate motional variation
of the p-ClPhꢀ spin adduct of the spin trap GS-PBN33
(Chart 1) in aqueous solutions containing various
concentrations of glycerol which can be used to modulate
the medium viscosity. As a comparison, similar studies were
also carried out on the p-ClPhꢀ adduct of PBN and a spin label
TEMPOL. In addition, a new phosphorylated derivative of
GS-PBN (GS-PPN, Chart 1) was synthesized which exhibits
Fig. 1 (A) EPR spectra of the nitroxide adducts GS-PBN–ClPh and
PBN–ClPh in aqueous solutions containing various concentrations of
glycerol (v/v). (B) Plot of the tC values of GS-PBN–ClPh, PBN–ClPh
and TEMPOL as a function of viscosity. The nitroxide adducts
GS-PBN–ClPh and PBN–ClPh were generated by UV photolysis of
aqueous solutions containing para-chlorophenyl diazonium tetrafluoro-
borate (8 mM) and the spin trap GS-PBN (50 mM) or PBN (25 mM). See
more details in Experimental section.
improved O2 ꢁ-trapping properties than GS-PBN. The spin
ꢀ
trapping properties of GS-PPN and the sensitivity of its spin
adducts towards the medium viscosity were explored. Finally,
ꢁ
ꢀ
the location of the O2
adduct of GS-PPN in the PSII
membranes was estimated according to the plot of the
rotational correlation time of the adduct as a function of the
medium viscosity.
glycerol (50%) although it is much broader and weaker than
the other two doublets (Fig. 1A, right).
In order to quantitatively describe the molecular motions of
GS-PBN–ClPh in the presence of different concentrations of
glycerol, we calculated their rotational correlation times (tC)
by using the formula:
Results and discussion
EPR spectral sensitivity of the p-ClPhꢀ spin adduct of GS-PBN
1/2
tC = 6.5 ꢂ 10ꢁ10W0[(h0/h
)
ꢁ1
ꢁ 1] s
(1)
to the medium viscosity
In order to prove the concept that the small radical adduct of
GS-PBN can be used to report the medium environment, we
firstly investigated EPR spectra of its p-chlorophenyl radical
(p-ClPhꢀ) adduct (GS-PBN–ClPh) in the glycerol–water
system. The radical p-ClPhꢀ is chosen since its production
can be easily controlled by UV photolysis of para-chlorophenyl
diazonium tetrafluoroborate. As shown in Fig. 1A (left, top),
the EPR spectrum of GS-PBN–ClPh in aqueous solution
exhibits well-resolved six lines due to the hyperfine interaction
where h0 and h are the amplitudes of the central and high-
ꢁ1
field doublets, respectively, and W0 the peak to peak linewidth
of the central doublet (note: the factor 6.5 ꢂ 10ꢁ10 may slightly
vary with spin adducts of various kinds of free radicals).37
Fig. 1B shows the plot of the tC values of the nitroxide adducts
GS-PBN–ClPh and PBN–ClPh as well as the spin label
TEMPOL as a function of the medium viscosity. For each
nitroxide, the tC value increases with the medium viscosity and
the plot can be well fitted with the modified Stokes–Einstein
equation:
with the nitrogen (aN = 1.60 mT) and b-hydrogen (aH
=
0.43 mT).33 Careful inspection found that the spectrum is
asymmetric with a weaker and broader high-field doublet as
compared to the other two due to the anisotropy of the
nitrogen coupling. Exogenous addition of the viscous glycerol
broadens EPR lines because of the slower molecular tumbling,
and in the presence of 50% glycerol (v/v) the high-field doublet
could not be resolved (Fig. 1A, left). Comparatively, the EPR
spectrum of the p-ClPhꢀ spin adduct of PBN shows less
sensitive response to the medium viscosity. The high-field
doublet is still well-resolved even at high concentration of
tC/e = (kV/kBT)Z + t0
(2)
where tC is the rotational correlation time, e the correction
factor of the rotational correlation time, Z the solvent viscos-
ity, k a dimensionless interaction parameter, V the van der
Waals volume of the rotating molecule, kB Boltzmann’s con-
stant, T the absolute temperature and t0 the zero viscosity
rotational correlation time.38 Due to the linkage of the bulky
glutathionyl group, GS-PBN–ClPh has a bigger van der Waals
c
1486 New J. Chem., 2011, 35, 1485–1490
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volume and thus affords a bigger slope (kV/kBT) [(2.04 ꢃ 0.06) ꢂ
10ꢁ10 s pꢁ1] as compared to PBN–ClPh [(0.49 ꢃ 0.02) ꢂ
10ꢁ10 s pꢁ1] and TEMPOL [(0.12 ꢃ 0.003) ꢂ 10ꢁ10 s pꢁ1]. It
can be therefore expected that the conjugation of a relatively
bulky group with the nitrone spin trap is helpful to increase the
sensitivity of its spin adducts to the medium viscosity.
those of its analogues17,31,42 but much longer than that of the
superoxide spin adduct of PBN (t1/2 { 1 min).39
In addition, GS-PPN can also be used to trap other oxygen,
carbon, and sulfur-centered radicals. Table S1 lists a summary
of all the observed EPR spectral parameters for these spin
adducts (see ESIw). Different from the EPR spectra of
GS-PBN spin adducts where six EPR lines were observed
due to the hyperfine interaction with the nitrogen and
b-hydrogen, the EPR spectra of GS-PPN spin adducts consist
of 12 lines owing to the additional phosphorus coupling (Table
S1, ESIw). The presence of the phosphorus coupling enables
more effective characterization of the trapped radicals as
suggestedꢁin previous studies.17,31 Interestingly, in the case of
Spin trapping of various reactive radicals by GS-PPN
ꢁ
ꢀ
The detection of O2 by the glutathione-linked PBN analogue
(GS-PBN) was limited due to the short half-life of thꢁe
ꢁ
corresponding O2 adduct.39 In order to improve the O2
ꢀ
ꢀ
detection, we designed a new phosphorylated derivative of
GS-PBN (GS-PPN, Chart 1) according to the recent
experimental and theoretical results.17,40,41 The synthetic
procedure of GS-PPN is shown in Scheme 1 (see more details
in Experimental section).
ꢀ
the SO3 spin adduct (Fig. 2b), only six EPR lines were
observed perhaps due to the small aH (B0.1 mT). This
relatively small aH could be due to the strong electron-
withdrawing property of the sulfite group. Similar results were
also observed from other spin traps.24,43,44 Therefore, the
ꢁ
ꢀ
Fig. 2a shows the EPR spectrum of the O2 adduct of
GS-PPN (GS-PPN–OOH) generated using xanthine/
a
introduction of the phosphoryl groupꢁinto the spin trap not
ꢀ
xanthine oxidase (X/XO) system. The spectrum can be
unambiguously assigned to GS-PPN–OOH as supported by
its similarity to the spectra of its analogues PPN17 and
LAMPPN,31 and suppression of the EPR signal in the
presence of SOD (400 U mlꢁ1) (figure not shown). As shown
in Fig. 2a, the EPR spectrum of GS-PPN–OOH consists of
12 lines due to the hyperfine interaction with the nitrogen
(aN = 1.36 mT), b-hydrogen (aH = 0.22 mT), and phosphorus
(aP = 4.23 mT). A kinetic study showed that GS-PPN–OOH
had a half-life time of 3.4 min (see ESIw), which is similar to
only significantly enhances the O2
detection but also
provides additional phosphorus coupling for its spin adducts
which is useful for the characterization of the trapped radicals.
Comparing the sC values of GS-PBN and GS-PPN spin adducts
Similar to the spin adducts of GS-PBN, the GS-PPN adducts
also exhibit asymmetric EPR spectra with the broad high-field
doublets at either half of the spectrum (due to the large
phosphorus splitting). The tC values for ten common spin
adducts are summarized in Table 1. To explore whether the
bulky glutathionyl group in GS-PPN spin adducts plays a
significant role in the spectral anisotropy, the tC values of
GS-PBN spin adducts were also determined (Table 1). The
same spin adducts of GS-PPN and GS-PBN gave similar tC
values, implying that the bulky glutathionyl group primarily
affects their rotational motions in solutions and offsets minor
effects of their structural differences, consistent with the result
as mentioned above. The charged and steric nature of the
trapped radicals has an influence on the tC values for the spin
adducts of either spin trap (Table 1). For example, in the case
of a-hydroxylalkyl radical adducts, the tC value increases from
the primary to tertiary hydroxylalkyl radical with the big tC
for the (CH3)2CꢀOH spin adduct (tC = 5.33 ꢂ 10ꢁ10 s for
GS-PPN and 6.32 ꢂ 10ꢁ10 s for GS-PBN) and the small one
Scheme 1 Synthesis of the glutathione-linked nitrone GS-PPN;
(i) diethyl (1-hydroxyamino-1-methylethyl) phosphonate, reflux,
chloroform, 91%; (ii) sodium iodide, reflux, ethanol, 61%; (iii)
glutathione, room temperature, phosphate buffer–ethanol (1/3 v/v),
quantitative.
for the CH2 OH spin adduct (2.88 ꢂ 10ꢁ10 s for GS-PPN and
ꢀ
2.45 ꢂ 10ꢁ10 s for GS-PBN). Interestingly, the charged O2
,
ꢁ
ꢀ
ꢁ
2ꢁ
ꢀ
ꢀ
CO2 and SO3 spin adducts show the relatively small tC
Table 1 Rotational correlation time tC (ꢂ10ꢁ10 s) of GS-PPN and
GS-PBN spin adducts in aqueous solutions
a
b
a
b
tC
Adduct
tC
tC
Adduct
tC
ꢁ
ꢀ
O2
1.98c
3.36d
2.67e
2.67e
1.76
CH2ꢀOH
2.88
3.79
5.33
4.21
2.77
5.47e
2.45
3.78
6.32
4.07
3.01
6.04e
CH3CHꢀOH
(CH3)2CꢀOH
C3H7CHꢀOH
ClPhꢀ
Fig. 2 EPR spectra of GS-PPN spin adducts. (a) O2 ꢁ; the adduct
ꢀ
CH3Oꢀ
EtOꢀꢀ
2.48e
2.37e
1.97
2.13
was obtained by addition of xanthine oxidase (0.02 U mlꢁ1) to a
ꢁ
reaction mixture containing GS-PPN (50 mM), xanthine (0.4 mM) and
ꢁ
CO2
ꢀSO3
ꢀ
2ꢁ
DTPA (0.1 mM) in an oxygenated PBS (20 mM, pH 7.4); (b) SO3
;
1.69
the adduct was generated by adding FeSO4 (0.1 mM) to the reaction
mixture containing GS-PPN (25 mM), H2O2 (2 mM) and EDTA
(2 mM) in the presence of Na2SO3 (0.2 M).
a
b
GS-PPN spin adducts. GS-PBN spin adducts. In the X/XO
c
d
system. In photosystem II. In DMSO.
e
c
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values. Therefore, like the hyperfine splitting constant, the tC
value could be the other important parameter for the
characterization of the trapped radicals.
the local environment of GS-PPN–OOH in the PSII membrane
was determined to be similar to that of the aqueous solution
containing B15% glycerol (Z E 1.33 p). The slightly
immobilized motion of GS-PPN–OOH in the PSII indicates
its weak interaction with the PSII membranes. Note: the
relatively high O2 concentration in the membrane due to
improved O2 solubility in the hydrophobic area can also result
in the EPR line broadening, therefore leading to slight
overestimation of the tC value measured in the PSII membrane.
Since both GS-PPN and its spin adduct GS-PPN–OOH are
amphiphilic consisting of the lipophilic aromatic moiety and a
hydrophilic glutathionyl group, it can be hypothesized that
GS-PPN is firstly situated on the surface of the membrane
ꢁ
ꢀ
Estimating the local environment of the O2 adduct of
GS-PPN in the PSII membranes
To test if the spin adducts of GS-PPN can also be used to
report their molecular rotational motions, we investigated the
tC variation of the p-ClPhꢀ spin adduct of GS-PPN with the
medium viscosity. Fig. 3A shows EPR spectra of the adduct in
the presence of various concentrations of glycerol. According
to these EPR spectra, the tC values were determined and
found to be well linear with the medium viscosity (Fig. 3B),
indicating that the adduct has a good sensitivity to the medium
viscosity.
ꢁ
ꢀ
and then trap the O2
illumination.11
from the PSII membranes after
In order to further demonstrate the potential application of
GS-PPNꢁas a dual free radical probe, we utilized GS-PPN to
Conclusions
ꢀ
trap O2 generated in the photosystem II (PSII) of spinach
The spin adducts of two glutathione-linked spin traps
(GS-PPN and GS-PBN) are highly sensitive to the medium
viscosity as compared to the corresponding spin adduct of
PBN and the spin label TEMPOL. In addition, various spin
adducts of both spin traps afford different tC values, therefore
implying that the tC value, like hyperfine splitting constant,
may act as the other important parameter for the character-
ization of the trappedꢀradical. The newly synthesized GS-PPN
exhibits improved O2 ꢁ-spin trapping properties with a half-
life time of 3.4 min in PBS. The biological application of
GS-PPN as a dual free radical probeꢁhas been satisfactorily
chloroplasts. Irradiation of the solution containing PSII
membranes and GS-PPN yielded a pure and intense signal
of GS-PPN–OOH (Fig. 3B, inset). The obtained hyperfine
splitting constants in this system (aP = 4.23 mT, aN
1.35 mT and aH = 0.22 mT) were well consistent with the
values in a standard X/XOD system (aP = 4.23 mT, aN
=
=
1.36 mT and aH = 0.22 mT, see Table S1 (ESIw)). However,
the EPR spectrum of GS-PPN–OOH in the PSII membrane
has a slightly stronger anisotropic broadening (tC = 3.36 ꢂ
10ꢁ10 s) than that in PBS (tC = 1.98 ꢂ 10ꢁ10 s, Table 1),
indicative of the slower rotational motions of the adduct in the
presence of the PSII membranes. Based on the plot of the tC
values as a function of the medium viscosity (Fig. 3B),
ꢀ
demonstrated by the detection of O2 generated in the PSII
membranes of chloroplasts. The present study opens up a new
possibility using the spin trapping technique to simultaneously
identify free radicals and determine the location of their spin
adducts in biological systems.
Experimental section
Reagents
Xanthine (X), xanthine oxidase (XO), superoxide dismutase
(SOD; from Bovine Erythrocytes) and 4-hydroxy-2,2,6,6-
tetramethylpiperidine-N-oxyl (TEMPOL) were used as
purchased from Sigma. Diethylenetriaminepentaacetic acid
(DTPA) was purchased from TCI. All solvents and reagents
were purchased without further purification. Double-distilled
water was used, and phosphate buffer solution (0.2 M, pH 7.4)
was stirred overnight in the presence of a chelating iminodiacetic
acid resin (4 g lꢁ1) to remove trace metal impurities.
Synthesis
The synthetic procedure of GS-PPN is shown in Scheme 1.
Diethyl [1-methyl-1-[4-(iodoacetamino) benzylidene] azinoylethyl]
phosphonate, 2, was obtained by condensation of 4-(chloro-
acetamido) benzaldehyde 133 and diethyl (1-hydroxyamino-1-
methylethyl) phosphonate,45 followed by iodination using
sodium iodide. Thereafter, the coupling reaction between
the compound 2 and GSH led to the glutathione-linked
phosphorylated nitrone GS-PPN that was well characterized
Fig. 3 (A) EPR spectra of spin adducts GS-PPN–ClPh in aqueous
solutions containing (a) 0%, (b) 10%, (c) 30% and (d) 50% glycerol
(v/v). (B) Plot of the tC values of GS-PPN–ClPh and GS-PPN–OOH
as
a function of viscosity; inset shows the EPR spectrum of
1
GS-PPN–OOH. See more details in Experimental section.
by H-, 13C-NMR and MS.
c
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1488 New J. Chem., 2011, 35, 1485–1490

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Synthesis of compound 1. Under a nitrogen atmosphere,
diethyl (1-hydroxyamino-1-methylethyl) phosphonate (0.40 g,
2 mmol) and 4-(chloroacetamino) benzaldehyde (0.47 g,
2.2 mmol) were dissolved in 30 ml of dry chloroform. The
resulting reaction mixture was refluxed for 4 h and then
filtered under reduced pressure. The solvent was evaporated
in vacuo and the resulting crude product was recrystallized
from ethanol–ether to afford a white solid (0.72 g, 1.8 mmol,
91%). 1H NMR (CD3OD): d 1.356 (6H, t), 1.847 (6H, d,
15.3 Hz), 4.204 (2H, s), 4.234 (4H, m), 7.730 (2H, d, J =
8.8 Hz), 7.906 (H, s), 8.353 (2H, d, J = 8.8 Hz). 13C NMR
(CD3OD): d 16.72 (d, 22.2 Hz), 23.53, 44.09, 64.92 (d,
27.6 Hz), 74.51 (d, 634.8 Hz), 120.46, 127.83, 131.74, 135.59,
141.90, 167.53. 31P NMR (CD3OD, H3PO4, external
Preparation of PSII membranes
PSII membranes from spinach were isolated as previously
described with minor modifications.46 Samples were resuspended
in the SMN buffer containing 0.4 M sucrose, 50 mM
MES–NaOH (pH 6.0), 10 mM NaCl, and stored at ꢁ80 1C.
Chlorophyll concentration was determined according to
Arnon’s method.47 For EPR measurements, the PSII
membranes were diluted to a concentration of 2.5 mg
Chl mlꢁ1 in buffer solution.
EPR measurements
All EPR measurements were performed using a Bruker ESP
300 EPR spectrometer operating at the X-band. EPR samples
were transferred in a quartz flat cell. Typical spectrometer
settings were microwave frequency, 9.5 GHz; modulation
amplitude, 0.05–0.15 mT; modulation frequency, 100 kHz;
microwave power, 12.9 mW; conversion time, 82 ms; time
constant, 164 ms; and receiver gain, 104–106. UV photolysis
was performed using a 200 W high-pressure mercury lamp.
Long-wave photolysis was performed by a continuous He–Ne
laser (25 mW, 663 nm) that produced strong irradiation
without heating in 5 min.
35
standard): d 24.07. MS (ESI, m/z): 391.1 ([MCl + H]+,
37
35
100%), 393.1 ([MCl + H]+, 28.6), 413.1 ([MCl + Na]+,
15.4), 428.9 ([MCl35 + K]+, 69.2), 431.1 ([MCl37 + K]+, 44.0).
Synthesis of compound 2. Diethyl [1-methyl-1-[4-(chloro-
acetamino)benzylidene]azinoylethyl] phosphonate (0.36 g,
0.92 mmol) and sodium iodine (0.69 g, 4.6 mmol) were
dissolved in 30 ml of ethanol and refluxed for 80 h in darkness.
The reaction mixture was filtered and concentrated in vacuo.
The resulting residue was dissolved in 100 ml of ethyl acetate
and washed with 3 ꢂ 100 ml of water. The organic layer was
dried on anhydrous Na2SO4, filtered and concentrated. A
white solid was obtained (0.27 g, 0.56 mmol, 61%). The
product was further purified by recrystallization in ethanol.
1H NMR (DMSO-d6): d 1.216 (6H, t), 1.695 (6H, d, 14.7 Hz),
4.068 (4H, m), 4.266 (2H, s), 7.650 (2H, d, J = 7.3 Hz), 7.836
(H, s), 8.317 (2H, d, J = 7.6 Hz), 10.548 (H, s). 13C NMR
(DMSO-d6): d 16.25, 22.85, 43.54, 62.62, 72.55 (d, 618 Hz),
118.57, 126.54, 129.56, 131.22, 139.76, 164.86. 31P NMR
(CD3OD, H3PO4, external standard): d 27.92. MS (ESI,
m/z): 483.1 ([M + H]+, 100%).
EPR spin trapping studies
ꢁ
ꢀ
(a) Spin trapping of O2
from the X/XO system. The
ꢁ
ꢀ
O2 adduct (GS-PPN–OOH) was generated using xanthine
(0.4 mM) and XO (0.02 U mlꢁ1) in the presence of GS-PPN
(50 mM) and DTPA (0.1 mM) in PBS (pH 7.4, 20 mM).
(b) Spin trapping of para-chlorophenyl radical (p-ClPhꢀ).
The radical was generated by UV irradiation of a solution
containing para-chlorophenyl diazonium tetrafluoroborate
(8 mM) and GS-PPN (50 mM) in DMSO or PBS (pH 7.4,
20 mM).
(c) Spin trapping of miscellaneous radicals. a-Hydroxylalkyl
radicals, CO2 ꢁ and SO3 ꢁ were generated from the solutions
containing H2O2 (2 mM), GS-PPN (50 mM), FeSO4 (0.1 mM)
and ethylenediaminetetraaceticacid (EDTA, 2 mM) in the
presence of alcohol (10% v/v of methanol, ethanol, isopropanol
or n-butanol), HCO2Na (0.2 M) and Na2SO3 (0.2 M),
respectively.
ꢀ
ꢀ
Synthesis of GS-PPN. Compound 2 (0.20 g, 0.41 mmol) in
30 ml of ethanol was mixed with a solution of glutathione
(0.10 g, 0.33 mmol) in 10 ml of K-phosphate buffer (0.1 M,
pH 7.4). The reaction mixture was stirred overnight at 25 1C in
darkness and concentrated to a volume of 5 ml. Then 10 ml of
chloroform was added to the reaction mixture. The resulting
solution was shaken and the lower phase was removed. The
procedure was repeated about 6 times until, assessed by thin
layer chromatography, the nitrone 2 in the chloroform
disappeared. The aqueous phase was concentrated in vacuo
and purified by Sephadex LH-20, eluting with water.
The colorless or pale yellow solid was obtained (0.22 g,
(d) Formation of alkoxy radical adducts. The spin adducts
of methoxy and ethoxy radicals were produced by adding a
small amount of Pb(OAc)4 to a solution of GS-PPN (50 mM)
and the corresponding alcohol (10%, v/v) in DMSO.
Acknowledgements
The research was supported by NSFC (No. 20875093 and No.
90813021) and the Pilot Project of Knowledge Innovation
Program of the Chinese Academy of Sciences.
1
quantitative). H NMR (D2O): d 1.256 (6H, t), 1.774 (6H, d,
J = 15.65 Hz), 1.903 (2H, m), 2.312 (2H, t), 2.926 (1H, m),
3.122 (1H, m), 3.257 (1H, t), 3.687 (2H, m), 4.178 (4H, m),
4.558 (1H, t), 4.736 (2H, s), 7.585 (2H, d, J = 8.5 Hz), 7.918
(1H, s), 8.211 (2H, d, J = 8.5 Hz). 13C NMR (D2O): d 17.13,
23.55, 31.34, 33.56, 35.08, 44.88, 54.34, 56.73, 66.56, 73.55,
74.60, 122.14, 127.21, 132.76, 139.37, 141.67, 172.50, 173.23,
177.51, 177.67, 182.20. 31P NMR (D2O, H3PO4, external
standard): d 25.06. MS (ESI, m/z): 662.1 ([M + H]+,
100%), 700.1 ([M + K]+, 47.3%).
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