6
a
0
.05 mmHg, 2.9 g (48%). IR (CHCl ) 1740 (C᎐O), 1585
᎐
Translation (Marlboro, MA) DT2721 computer interface
3
᎐
Ϫ1
(
C᎐N–O) cm .
board and locally written acquisition software. Data for signal
height versus dose were obtained from multiple measurements
on samples exposed to given radiation doses as a function of
time after radiation. These were extrapolated to the signal
height at the end of radiation using a linear exponential
regression. Error estimates on the signal height versus time
scans derived from two sources. The first was the standard
signal-to-noise (S/N). A portion of the baseline representing
approximately 1/10 the spectral scan was selected as typical.
Peak-to-peak deviation was measured. The ratio of this signal
to the interval was multiplied by 3 to represent the signal to
RMS noise. A second source of measurement uncertainty was
the insertion error. This is estimated to be 6% based on the
reinsertion of stable sample into the cavity. The final errors
reflect the larger of the two sources.
14
5
-Carboxy-5-methyl-1-pyrroline N-oxide (2) and 5-carboxy-
15
5
1
-methyl-1-pyrroline N-oxide (7). 5-Ethoxycarbonyl-5-methyl-
-pyrroline N-oxide (2 g) was hydrolyzed by treatment with an
aqueous solution of sodium hydroxide (2.5%, 20 mL) at 100 ЊC
for 1 h. Upon cooling, this solution was passed through a
column containing Dowex 50 (H form, Biorad, Richmond,
CA). Fractions were collected and rotary evaporated under
vacuum to dryness, giving 5-carboxy-5-methyl-1-pyrroline N-
oxide 2. Crude nitrone 2 was purified by flash chromatography
using silica gel (mesh 230–400). By eluting the column with
chloroform–methanol (11 : 1), 5-carboxy-5-methyl-1-pyrroline
N-oxide 2 (1.2 g, 70%) was obtained as a white solid, a portion
+
6a
of which was recrystallized from CHCl , mp 135–136 ЊC. IR
CHCl ) ν: 3500–3300 (O–H), 1715 (C᎐O), 1585 (C᎐N–O) cm ;
3
Ϫ1
(
3
NMR (D O) δ: 1.60 (s, 3H), 2.10–2.30 (m, 1H), 2.40–2.65 (m,
Spin trapping superoxide
2
1
H), 2.65–2.90 (m, 2H), 7.35 (s, 1H); m/z 143.
2ؒ
Ϫ
15
Spin trapping of O
by nitrone 2 (60 mM) and nitrone 10
5
-Carboxy-5-methyl-1-pyrroline1 N-oxide 7 was prepared as
5
(60 mM) was achieved by mixing the appropriate reactants as
described above. All spectra were recorded at room temperature
using an EPR spectrometer (Varian Associates E-9). Reaction
mixtures were transferred to a flat quartz cell and fitted into the
cavity of the spectrometer. Instrumentation settings were:
microwave power, 20 mW; modulation frequency, 100 kHz;
described above, except sodium [ N]nitrite was used in place
of sodium [ N]nitrite in the first step. Nitrone 7 was isolated
as a white solid, mp 135–136 ЊC (from CHCl ). IR (CHCl )
ν: 3500–3300 (O–H), 1715 (C᎐O), 1585 (C᎐N–O) cm ; NMR
14
6a
3
3
Ϫ1
᎐
᎐
(
D O) δ: 1.60 (s, 3H), 2.10–2.30 (m, 1H), 2.40–2.65 (m, 1H),
2
2
.65–2.90 (m, 2H), 7.35 (s, 1H); m/z 143.9.
modulation amplitude, 1.0 G; response time, 1 s; and sweep,
Ϫ1
1
2.5 G min .
Generation of superoxide
ؒ
Superoxide was generated using NADPH and FMN at pH 7.4
Rate of the spin trapping of HO
16
ؒ
Ϫ
and 1.95. Specifically, O2 production was achieved by
mixing NADPH (1 mM) and FMN (1 mM) in sodium phos-
phate buffer (50 mM) containing DTPA (1 mM), at pH 7.4 and
KCl (50 mM and HCl solution titrated to pH 1.95 with a
NaOH solution) containing DTPA (1 mM).
To determine the apparent rate constant for the spin trapping
of HO by 5-carboxy-5-methyl-1-pyrroline N-oxide 2, the mix-
ture of nitrone 2 (60 mM), H O (17.6 mM) and nitrone 1 (0 –
0 mM) were photolyzed for 1 min, as previously described.
Reaction mixtures were immediately transferred to an EPR
flat quartz cell and introduced into the cavity of the EPR
spectrometer. EPR spectra were recorded 1 min after the
termination of the photolysis.
ؒ
2
2
1
3
6
Spin trapping of hydroxyl radical
ؒ
Photolysis of H O was used as a source of HO . A solution of
2
2
14
either 5-carboxy-5-methyl-1-pyrroline N-oxide 2 (60 mM) or
-carboxy-5-methyl-1-pyrroline N-oxide 7 (60 mM), contain-
15
5
ؒ
Stability of the spin trapped adduct of HO
ing H O (17.6 mM) was irradiated by exposing this reaction to
2
2
ؒ
The stability of the spin trapped adduct of HO was estimated
using methods detailed earlier. In a typical experiment, 1 mL of
-carboxy-5-methyl-1-pyrroline N-oxide 2 (40 mM) in sodium
phosphate buffer (50 mM) at pH 7.4 containing DTPA (1 mM)
UV light (Ultra-Violet Products, Inc, San Gabriel, CA, SCTI
model) for 1 min in sodium phosphate buffer (50 mM) at pH 7.4
containing DTPA (1 mM). EPR Spectra were recorded 1 min
after termination of photolysis. The Fenton reaction was used
5
Ϫ1
ؒ
was irradiated with 10 Gy at a dose rate of 2.12 Gy min
as an alternative source of HO by mixing H O (17.6 mM) and
2
2
2+
(PANTAK pmc1000, 150kVp, 25 mA, 1.6 mm Al filter,
HVL = 1.9 mm Cu). Approximately 2 min after irradiation was
completed, the sample was transferred to a quartz TM flat cell
and fitted into the cavity of a Varian E-12 EPR spectrometer.
EPR spectra were recorded at room temperature at various time
intervals until the peak height decreased to several half-lives.
Fe (80 µM) in phosphate buffer at pH 7.4. All spectra were
recorded at room temperature using an EPR spectrometer
(
Varian Associates E-9 or E-109). Reaction mixtures were
transferred to a flat quartz cell and fitted into the cavity of the
EPR spectrometer. Instrumentation settings were: microwave
power, 20 mW; modulation frequency, 100 kHz; modulation
Ϫ1
amplitude, 1.0 G; response time, 1 s; and sweep, 12.5 G min .
Modeling of EPR spectra
X-Ray irradiation of H O was used as an alternative source
2
ؒ
of HO . 5-Carboxy-5-methyl-1-pyrroline N-oxide 2 (1 mL, 40
EPR spectra were modeled using the Bruker Symphonia and
Bruker WinEPR programs.
mM) in sodium phosphate buffer (50 mM) at pH 7.4 containing
DTPA (1 mM) was irradiated with 1, 2, 5, 10 or 14.7 Gy at a
Ϫ1
dose rate of 2.12 Gy min (PANTAK pmc1000, 150kVp, 25
mA, 1.6 mm Al filter, half value layer (HVL) = 1.9 mm Cu).
Approximately 2 min after irradiation was completed, the
sample was transferred to a quartz TM flat cell and fitted into
the cavity of a Varian E-12 EPR spectrometer equipped with a
century series bridge (E-102) and with a TM011 cavity mounted
horizontally in the magnet. EPR spectra were recorded at
X-band (9.45 GHz), using 30 mW of power, with time constant
of 0.3 s, a dwell time of 0.3 s, 512 points per spectrum, (∼2.5
Acknowledgements
This research was supported in part by grants from the
National Institutes of Health, RR-12257, CA-69538 and T32-
ES07263.
References
5
1 W. C. Röntgen, Würzberger Physik.-Med. Ges., 1895, 9, 132.
E. H. Grubbé, Radiology, 1933, 21, 156.
J. Weiss, Nature, 1944, 153, 748.
H. J. Halpern, C. Yu, E. Barth, M. Peric and G. M. Rosen, Proc.
Natl. Acad. Sci. USA, 1995, 92, 796.
min per spectrum), receiver gain of 2 × 10 and modulation
2
3
4
amplitude of 0.5 G. Data were logged on a PC interfaced with
the Varian console via an interface built by R. Quine, University
of Denver, a set of 12 bit A/D and D/A channels on a Data
J. Chem. Soc., Perkin Trans. 2, 2001, 875–880
879