Synthesis of nitroxyl radicals for overhauser-enhanced magnetic resonance imaging

Article abstract of DOI:10.1002/ardp.200800053

Non-invasive measurement and visualization of free radicals in vivo would be important to clarify their roles in the pathogenesis of free radical-associated diseases. Nitroxyl radicals can react with free radicals and be derivatized to achieve specific cellular / subcellular localizing capabilities while retaining the simple spectral features useful in imaging. Overhauser-enhanced magnetic resonance imaging (OMRI), which is a double resonance technique, creates images of free radical distributions in small animals by enhancing the water proton signal intensity via the Overhauser Effect. In this study, we synthesized various nitroxyl probes having ¹⁵N nuclei and deuterium, and measured the enhancement factor for Overhauser-enhanced magnetic resonance imaging experiments. ¹⁵N-D-4- Oxo-2,2,6,6-tetramethylpiperidine-1-oxyl (¹⁵N-D-oxo-TEMPO) has the highest enhancement factor compared with other nitroxyl probes. The proton signal enhancement was higher for ¹⁵N-labeled nitroxyl probes when compared to the 14N-labeled analogues because of the reduced spectral multiplicity of the I = 1/2 nucleus. Furthermore, this enhancement is proportional to the line width and number of electron spin resonance lines of nitroxyl radicals. Finally, we compared the Overhauser-enhanced magnetic resonance image of ¹⁵N-labeled, deuterated 4-Oxo-2,2,6,6- tetramethylpiperidine-1-oxyl with that of ¹⁴N-H-TEMPOL. These results suggested that the selective deuteration of the nitroxyl probes enhanced the signal-to-noise ratio and thereby improved spatial and temporal resolutions.

Full text of DOI:10.1002/ardp.200800053

5
48
Arch. Pharm. Chem. Life Sci. 2008, 341, 548–553
Full Paper
Synthesis of Nitroxyl Radicals for Overhauser-enhanced
Magnetic Resonance Imaging
1
1
1
1
1
Ken-ichi Yamada , Yuichi Kinoshita , Toshihide Yamasaki , Hiromi Sadasue , Fumiya Mito , Mika
1
1
2
2
1
1
Nagai , Shingo Matsumoto , Mariko Aso , Hiroshi Suemune , Kiyoshi Sakai , and Hideo Utsumi
1
Department of Bio-function Science, Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka,
Japan
2
Department of Pharmaceutical Synthetic Chemistry, Faculty of Pharmaceutical Sciences, Kyushu University,
Fukuoka, Japan
Non-invasive measurement and visualization of free radicals in vivo would be important to clar-
ify their roles in the pathogenesis of free radical-associated diseases. Nitroxyl radicals can react
with free radicals and be derivatized to achieve specific cellular / subcellular localizing capabil-
ities while retaining the simple spectral features useful in imaging. Overhauser-enhanced mag-
netic resonance imaging (OMRI), which is a double resonance technique, creates images of free
radical distributions in small animals by enhancing the water proton signal intensity via the
1
5
Overhauser Effect. In this study, we synthesized various nitroxyl probes having N nuclei and
deuterium, and measured the enhancement factor for Overhauser-enhanced magnetic reso-
1
5
15
nance imaging experiments. N-D-4-Oxo-2,2,6,6-tetramethylpiperidine-1-oxyl ( N-D-oxo-TEMPO)
has the highest enhancement factor compared with other nitroxyl probes. The proton signal
15
14
enhancement was higher for N-labeled nitroxyl probes when compared to the N-labeled ana-
logues because of the reduced spectral multiplicity of the I = 1/2 nucleus. Furthermore, this
enhancement is proportional to the line width and number of electron spin resonance lines of
nitroxyl radicals. Finally, we compared the Overhauser-enhanced magnetic resonance image of
15
14
N-labeled, deuterated 4-Oxo-2,2,6,6-tetramethylpiperidine-1-oxyl with that of N-H-TEMPOL.
These results suggested that the selective deuteration of the nitroxyl probes enhanced the sig-
nal-to-noise ratio and thereby improved spatial and temporal resolutions.
Keywords: DNP / Free radical / Nitroxyl radicals / OMRI / Redox /
Received: March 6, 2008; accepted: May 5, 2008
DOI 10.1002/ardp.200800053
Introduction
each living cell is placed: namely, functional in physio-
logical conditions, but harmful in a variety of pathologi-
Free radicals including reactive oxygen and nitrogen spe- cal conditions. Free-radical reactions in vivo appear
cies reveal two completely different characteristics extremely complicated because there are numerous mol-
depending on the dose loaded and the situation in which ecules and reaction pathways that might influence radi-
cal reactions. Furthermore, the oxygen concentration in
most tissues in vivo is much lower than that in in-vitro
Correspondence: Hideo Utsumi Ph.D., Department of Bio-function Sci-
ence, Faculty of Pharmaceutical Sciences, Kyushu University, 3-1-1 Hi-
gashi-ku, Maidashi, Fukuoka 812-8582, Japan.
E-mail: utsumi@pch.phar.kyushu-u.ac.jp
experiments. Therefore, non-invasive measurement and
visualization of free radicals in vivo would be important
to clarify their roles in the pathogenesis of free radical-
associated diseases.
The most direct technique available for the detection
of reactive free radicals in vivo is electron spin resonance
Fax: +81-92-642-6626
Abbreviations: Electron spin resonance (ESR); dynamic nuclear polar-
ization (DNP); Overhauser-enhanced magnetic resonance imaging (OM-
RI); 4-Oxo-2,2,6,6-tetramethylpiperidine-1-oxyl (oxo-TEMPO); 4-hydrox-
yl-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL); triaryl-methyl (TAM)
(ESR) spectroscopy, which requires
a
spin-probe
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Arch. Pharm. Chem. Life Sci. 2008, 341, 548–553
Nitroxyl Radicals for OMRI
549
approach. The in vivo spin-probe technique has been used Results and discussion
to evaluate the generation of free radicals [1–3] and the
Chemistry
redox status [4–7] by monitoring the ESR signal loss. The
reaction of nitroxyl radicals as spin-probes with free radi-
cals is dependent on the basic structure of nitroxyl
probes such as piperidine or pyrrolidine. Furthermore,
nitroxyl probes can be derivatized to achieve specific cel-
lular / subcellular localizing capabilities while retaining
the simple spectral features useful in imaging. Recently,
we developed a non-invasive technique to evaluate the in
vivo free-radical generation both in and out of mem-
branes in living animals using several spin probes with
different physical properties [2, 8]. The involvement of
lipid-derived free radicals was reported recently during
the initiation of the nitrosamine metabolism [8].
Synthesis of 2,2,6,6-tetramethyl-piperidine-4-one 1 was
carried out according to the methods of a previous report
[16]. The isotope-labeled compound 1 was either deuter-
1
5
15
ated using acetone-d or N-labeled with N-ammonium
6
chloride. Isotope-labeled nitroxyl probes were synthe-
sized by using the isotope-labeled compound 1 in the
manner depicted in Scheme 1 [17–19].
Electron spin resonance measurements
1
4
The nitroxyl radical with a N nucleus exhibited an Elec-
tron spin resonance (ESR) spectrum that exhibited a
three-line spectrum characteristic of the unpaired elec-
tron interacting with the N nucleus (I = 1), whereas the
nitroxyl radical derivative with the nitrogen nucleus sub-
1
4
Overhauser-enhanced magnetic resonance imaging
(
OMRI) is a double resonance technique that uses the
1
5
stituted with N (I = ꢀ) exhibited an ESR spectrum with a
doublet with hyperfine splitting (data not shown). The
reinforcement of the signal intensity of 40–50% was seen
presence of paramagnetic agents to enhance the signal
intensity from nuclear spins via a process known as
dynamic nuclear polarization (DNP) or Overhauser Effect
1
5
14
in the N body in comparison with a N body by a
decrease in division number.
[
9–12]. In this phenomenon, the relatively stronger mag-
netic moment of the electron is utilized to enhance the
polarization of the nuclear spins, thereby enhancing
their signal. The unique advantage of this technique is
high spatial resolution of the image and short acquisi-
tion time. The significant contrast-to-noise ratio obtained
Regarding the deuterated nitroxyl probes, the ESR
spectrum-line width became narrow to 70% in compari-
son with non-deuterated nitroxyl probes (Table 1).
Because the gyromagnetic ratio of deuterium atom is
1
1
: 6 in comparison with hydrogen, the ESR spectrum-
by this technique at very low magnetic fields for H-based
line width of deuterated nitroxyl probes is narrow. When
the signal intensity of deuterated nitroxyl probes rein-
forced the shared portions, that line width became nar-
row.
MRI detection (l10 mT) compared to the routinely used
MRI systems that operate at fields l1 T makes OMRI
advantageous in obtaining physiological information.
Alecci et al. utilized nitroxyl radicals and successfully
obtained images of water protons in tissue in the vicinity
of the paramagnetic radical and so achieved in-vivo imag-
2
.3 OMRI experiments
1
4
Figures 1a and b show the corresponding DNP spectra of
ing [13]. More recently, we reported that the use of
N
1
4
15
15
N- and N-labeled nitroxyl probes obtained by detecting
the proton intensities after ESR irradiation at appropri-
and its isotropic compound N nuclei to label nitroxyl
radicals with OMRI, can visualize the reaction with ascor-
bic acid on the inner and outer membranes of liposomes
14
ate magnetic fields in the range of 5–10 mT. The N-
labeled nitroxyl radical has a N nucleus that exhibits an
ESR spectrum of three lines, characteristic of an unpaired
electron interacting with a N nucleus. The unequal cou-
pling constants, which are usually observed at low-fre-
quency measurements, are caused by breakdown of the
high-field approximation, known as the“Breit-Rabi”
1
4
[
14]. Unfortunately, the utilization of nitroxyl radicals
used as spin probes for OMRI experiments is restricted
due to a broad-line width compared with the triaryl-
methyl (TAM) radical. Grucker et al. reported that the
enhancement factor of deuterated 4-Hydroxyl-2,2,6,6-tet-
ramethylpiperidine-1-oxyl (TEMPOL) was higher than
that of TEMPOL using a DNP spectrometer [15]. Further-
1
4
1
5
15
effect. N-labeled nitroxyl probes have N nuclei substi-
tuted for N nuclei; these exhibit an ESR spectrum with a
1
4
15
more, they suggested that with the N-labeled and deu-
terated compound the higher enhancement factor can
1
4
15
doublet with hyperfine splitting. The N and N hyper-
fine coupling constants obtained from the DNP spectra
for the two nitroxyls are in close agreement with the cor-
responding hyperfine coupling constants measured in
their ESR spectra. Table 2 shows DNP signal enhance-
ment in the presence of various nitroxyl probes in phos-
phate-buffered saline solution (pH = 7.4). Oxo-TEMPO (4-
15
be obtained because of the N nuclei spin (I = 1/2) and the
small gyromagnetic ratio of deuterium.
The aim of this study was to synthesize the various
15
nitroxyl probes that have a N nucleus and deuterium,
and measure the enhancement factor to design the new
nitroxyl probes to improve the OMRI image resolution.
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Arch. Pharm. Chem. Life Sci. 2008, 341, 548–553
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15
Table 1. Line width (lT) of N- and N-labeled nitroxyl probes.
1
4
14
15
15
Nitroxyl Probes
N-H
N-D
N-H
N-D
Oxo-TEMPO
TEMPOL
Carbamoyl-PROXYL
Carboxy-PROXYL
MC-PROXYL
39.6 l 3.02
29.9 l 0.97
98.8 l 3.00
79.4 l 4.78
76.5 l 3.45
80.9 l 4.85
39.1 l 1.87
151.8 l 1.40
114.4 l 2.62
115.8 l 5.99
113.4 l 7.71
31.1 l 1.21
121.0 l 2.88
70.1 l 1.44
78.2 l 2.81
73.7 l 4.32
139.7 l 0.67
114.4 l 4.76
126.0 l 3.24
115.6 l 7.51
Nitroxyl probes (10 lM) were dissolved in 0.1 M phosphate-buffered saline (pH 7.4). All values represent the mean l SD (n = 3).
14
15
Table 2. Enhancement factor of N- and N-labeled nitroxyl probes.
1
4
14
15
15
Nitroxyl Probes
N-H
N-D
N-H
N-D
Oxo-TEMPO
TEMPOL
Carbamoyl-PROXYL
Carboxy-PROXYL
MC-PROXYL
11.4 l 0.75
6.5 l 0.67
7.3 l 0.28
7.1 l 0.22
7.1 l 0.28
11.6 l 0.09
8.1 l 0.20
9.8 l 0.09
10.3 l 0.30
9.3 l 0.29
20.6 l 0.17
13.1 l 0.28
15.4 l 0.62
15.6 l 1.22
15.8 l 0.44
23.7 l 0.78
16.1 l 0.87
20.5 l 0.61
22.0 l 1.14
20.6 l 0.11
Nitroxyl probes (2 mM) were dissolved in 0.1 M phosphate-buffered saline (pH 7.4). All values represent the mean l SD (n=3).
Figure 2. Overhauser-enhanced magnetic resonance images in
the presence of nitroxyl probes (2 mM). (a) The scheme of eight
14
phantom tubes. (b) Electron spin resonance OFF image. (c) N-
1
5
H-TEMPOL image. (d) N-D-oxo-TEMPO image. The inner
diameters of the eight phantom tubes were 7, 6, 5, 4, 3, 2, 1.5,
and 0.7 mm. Experimental conditions are described in Experi-
mental, Section 3.
Figure 1. Dynamic 1nuclear polarization spectra of nitroxyl
4
15
probes (2 mM). (a) N-labeled nitroxyl radical, (b) N-labeled
nitroxyl radical.
nitroxyl probes give higher enhancement factors for OMRI ex-
periments.
Oxo-2,2,6,6-tetramethylpiperidine-1-oxyl) has a higher
enhancement factor compared with the other nitroxyl
probes. The proton signal enhancement was higher for
To examine the image conditions under which
nitroxyl probes can be seen, a phantom containing a
N-labeled nitroxyl probes when compared to the N- nitroxyl probe was placed in several different tubes and
15
14
labeled analogues because of the reduced spectral multi-
plicity of the I = 1/2 nucleus. Furthermore, this enhance-
ment is proportional to the line width and number of
tested. Figure 2a shows the scheme of eight phantom
tubes with different sizes of inner diameters. Figure 2b
shows a phantom image that was collected in the absence
ESR lines of nitroxyl radicals (Tables 1 and 2). These of ESR irradiation yet in the presence of the nitroxyl
results are in agreement with a previous report [20] that
mentioned that the enhancement factor can be written
as:
probe; this image is characteristic of a magnetic reso-
nance image with poor signal-to-noise ratio (SNR). The
poor SNR and spatial resolution of the image are consis-
tent with the low magnetic field (15 mT) at which the MR
images were collected. Although the gross features of the
phantom could be recognized, the image resolution is
not sufficient to provide a sharply defined image of the
phantom. The magnetic resonance images were collected
after a period of ESR irradiation; examples are shown in
Figs. 2c and d. The use of nitroxyl probes facilitated sig-
1
re
E ¼ 1 ꢀ n
qfs
ð1Þ
rn
where r
e
and r
n
are the electronic and nuclear gyromagnetic ra-
tios, respectively. The other variables in Eq. (1) are q, the cou-
pling factor f, the leakage factor s, the saturation factor, and n,
the number of lines in the free radical's ESR spectrum. From the
1
5
equation and in this experiment, the N-labeled and deuterated
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Arch. Pharm. Chem. Life Sci. 2008, 341, 548–553
Nitroxyl Radicals for OMRI
551
PROXYL),
3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl
carbamoyl-PROXYL), 4-oxo-2,2,6,6-tetramethylpiperidine-N-oxyl
oxo-TEMPO), and 4-hydroxyl-2,2,6,6-tetramethylpiperidine-N-
(
(
oxyl (TEMPOL) were purchased from Aldrich (St. Louis, MO, USA).
All other reagents were all obtained from Wako Pure Chemical
Industries (Osaka, Japan). Isotope-labeled nitroxyl probes were
synthesized by using the isotope-labeled 2,2,6,6-tetramethylpi-
peridine-4-one according to Scheme 1.
2
,2,6,6-Tetramethylpiperidine-4-one (Triacetoneamine) 1
15
A mixture of ammonium chloride or N-ammonium chloride
37.4 mmol), acetone, or acetone-d (98.6 mmol), anhydrous
sodium carbonate (173 mmol), and magnesium oxide
432 mmol) was added to a 1 L round-bottomed flask. The flask
(
6
(
was capped with a rubber septum and wired; then, the reaction
mixture was heated in an oil-bath at 508C for three days. After
cooling, 20 mL of acetone was added to the reaction mixture
and the resulting solution was filtered. The recovered solid was
crushed into powder, washed with 15 mL of acetone, and was
then filtered with suction filtration. The combined filtrates
were evaporated and purified with silica gel column chromatog-
raphy. The resulting red liquid was solidified by chilling in a dry
ice / acetone bath. The solid product was recrystallized with hex-
ane to afford 97 mmol (26.0%) of a colorless crystal. Calculated
for C
9
H17NO: C, 69.19; H, 10.97; N, 8.96. Found: C, 68.13; H, 10.94;
N, 8.91.
Scheme 1. Synthesis of isotope-labeled nitroxyl probes.
4
-Oxo-2,2,6,6-tetramethylpiperidine-1-oxyl (Oxo-
TEMPO)
nificantly enhanced images. The resolution in the image
was ~0.7 mm. It was found that the image intensity of
A solution of 2,2,6,6-tetramethylpiperidine-4-one (97 mmol) and
sodium tungstate dihydrate (4.82 mmol) in 99.7 mL of water was
cooled to 58C and 22.3 mL of 30–35.5% hydrogen peroxide was
added with stirring. The temperature did not exceed 258C. The
suspension was stirred vigorously for 24 h at ambient tempera-
ture. Then, it was saturated with potassium carbonate and
extracted with ether. The ether phase was dried over magnesium
sulfate, filtered, and evaporated. The residue was recrystallized
from hexane to give 79.6 mmol (82.1%) of an orange product.
1
5
N-deuterated oxo-TEMPO, which has a narrow line
14
width, was much higher than that of N-H-4-hydroxyl-
14
2
,2,6,6-tetramethylpiperidine-N-oxyl ( N-H-TEMPOL).
In conclusion, these results suggested that the selective
deuteration of the nitroxyl probes enhanced the signal-
to-noise ratio and thereby improved spatial and temporal
resolutions. The OMRI technique is a powerful imaging
modality for use in small animal research to understand
the mechanism of free radical-related diseases. Progress
in this field is largely conditional on the design of new
nitroxyl probes.
This work was partially supported by Development of System
and Technology for Advanced Measurement and Analysis from
Japan Science and Technology Agency and Grant-in-Aid for
Young Scientist (A) No. 19689002 from Japan Society for the
Promotion of Science.
1
-Hydroxyl-4-oxo-2,2,6,6-tetramethylpiperidine
hydrochloride
Aqueous hydrochloric acid (37%, 7.65 mL) was added dropwise
at 58C to a stirred solution of 4-oxo-2,2,6,6-tetramethylpiperi-
dine-1-oxyl (79.6 mmol) in ethanol (11 mL). The reaction mixture
was stirred for 1 h at 248C. The solvent was removed in a rotating
evaporator and the residue was recrystallized from 2-propanol
to give 53.9 mmol (67.7%) of a white solid. Calculated for
9 2
C H18ClNO : C, 52.05; H, 8.74; N, 6.74. Found: C, 52.07; H, 8.71; N,
6.72.
The authors have declared no conflict of interest.
3
-Bromo-4-oxo-2,2,6,6-tetramethylpiperidine-1-oxyl
A solution of bromine (53.9 mmol) in chloroform (5 mL) was
added dropwise to a stirred solution of 1-hydroxyl-4-oxo-2,2,6,6-
tetramethylpiperidine hydrochloride (53.9 mmol) in chloro-
form (110 mL). After a clear solution was observed, to this solu-
tion was added with vigorous stirring a solution of sodium
nitrite (121 mmol) in water (118 mL). The mixture was stirred
for 30 min at 248C. The chloroform layer was separated, washed
with water, and dried with anhydrous magnesium sulfate. After
removal of the solvent in a rotating evaporator, the residue was
Experimental
Materials
1
5
6
Ammonium chloride ( N, 99%) and acetone-d were obtained
from Cambridge Isotope Laboratories, Inc. (Andover, MA, USA).
-carboxy-2,2,5,5-tetramethylpyrrolidine-N-oxyl (carboxy-
3
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552
K. Yamada et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 548–553
1
5
recrystallized from hexane to give 31.8 mmol (59%) of an orange
product. Calculated for C : C, 43.22; H, 6.04; N, 5.60.
Found: C, 43.17; H, 6.05; N, 5.67.
C
10
H
3
D
15 NO
3
: C, 55.47; H, 8.38; N, 6.47. Found: C, 51.92; H, 7.93;
9
H15BrNO
2
N, 5.88.
4
-Hydroxyl-2,2,6,6-tetramethylpiperidine-1-oxyl
3
-Carboxy-2,2,5,5-tetramethylpyrrolidine-1-oxyl
(TEMPOL)
4
NaBH (16.4 mmol) was added to a stirred solution of 4-oxo-
(
Carboxy-PROXYL)
Compound 3-bromo-4-oxo-2,2,6,6-tetramethylpiperidine-1-oxyl
2
,2,6,6-tetramethylpiperidine-1-oxyl (65.6 mmol) in ethanol. The
reaction mixture was stirred for 1 h in an ice bath. Brine was
added to the solution, which was then extracted with ether. The
ether layer was dried with anhydrous magnesium sulfate. After
removal of the solvent in a rotating evaporator, the residue was
first purified with silica gel column chromatography, and then
recrystallized from a mixture of ether and hexane (2 / 1, v / v) to
give an orange product.
(
31.8 mmol) was added, portionwise, to a stirred solution of
potassium hydroxide (79.5 mmol) in water (79.5 mL). The reac-
tion mixture was stirred for 2 h at 248C, acidified with 5 N aque-
ous hydrochloric acid solution to pH = 3, and quickly extracted
with chloroform. The organic layer was dried with anhydrous
magnesium sulfate. After removal of the solvent in a rotating
evaporator, the residue was first purified with silica gel column
chromatography and then recrystallized from a mixture of
chloroform and hexane (3 / 1, v / v) to give 14.95 mmol (47%) of a
yellow product.
1
4
– 1
[
9 2 2
N, D] IR (cm ): 3413 (-OH), Calculated for C H D16NO : C,
1
5
5
7.34; H, 9.63; N, 7.44. Found: C, 59.31; H, 10.06; N, 7.57. [ N, H]
IR (cm ): 3411 (-OH), Calculated for C
10.47; N, 8.08. Found: C, 62.47; H, 10.52; N, 8.14. [ N, D] IR (cm
: 3413 (-OH), Calculated for C
–
1
15
9
H
18 NO
2
1
: C, 62.34; H,
5
–
1
4
– 1
[
N, D] IR (cm ): 2975 (-COOH), 1731 (C=O), Calculated for
1
15
)
9
H
2
D
16 NO
2
: C, 57.04; H, 9.57; N,
C HD15NO : C, 53.65; H, 8.00; N, 6.96. Found: C, 55.07; H, 8.23; N,
7
C
7
9 3
1
5
-1
7.39. Found: C, 58.98; H, 9.99; N, 7.62.
.11. [ N, H] IR (cm ): 2976 (-COOH), 1728 (C=O), Calculated for
1
5
9 3
H16 NO : C, 57.69; H, 8.61; N, 7.48. Found: C, 57.68; H, 8.53; N,
1
5
-1
.53. [ N,D] IR (cm ): 2973 (-COOH), 1730 (C=O), Calculated for
ESR and OMRI experiments
1
5
C
9
HD15 NO
3
: C, 53.38; H, 7.96; N, 6.92. Found: C, 54.57; H, 8.21;
The OMRI experiments were performed on a custom-built whole
body scanner (Philips Research Laboratories, Hamburg, Ger-
many) operating in a field-cycled mode to avoid excess power
deposition during the ESR cycle. The nuclear magnetic reso-
nance (NMR) field strength of the scanner is 15 mT and the NMR
transmission chain operates at a frequency of 625 kHz using a
saddle transmission coil. The receiving coil is a solenoid coil
tuned to 625 kHz with a band-width of 80 kHz. The maximum
transmission power is 250 W (peak). The ESR irradiation fre-
quency is 226 MHz and a saddle coil is used for the transmission.
N, 7.16.
3
-Carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl
Carbamoyl-PROXYL)
(
Portionwise, 3-bromo-4-oxo-2,2,6,6-tetramethylpiperidine-1-oxyl
(
(
10.6 mmol) was added to a solution of potassium hydroxide
31.0 mmol) in 29% ammonia (85.3 mL). The reaction mixture
was stirred for 1 h at 248C, and then extracted with chloroform.
The chloroform layer was dried with anhydrous magnesium sul-
fate. After removal of the solvent in a rotating evaporator, the
residue was first purified with silica gel column chromatogra-
phy, and then recrystallized from acetone to give 4.24 mmol
1
4
15
The corresponding DNP spectra of N- and N-labeled nitroxyl
radicals were obtained by detecting the proton intensities after
ESR irradiation in different magnetic fields in the range of 5–
10 mT spanning the ESR spectra of the nitroxyl radicals.
Enhancement factors of nitroxyl radicals were obtained from
the image intensities divided by ESR non-irradiated (ESR OFF)
image intensities. Enhancement factors were then correlated
with the ESR line-widths of each nitroxyl radical. The ESR line-
width was measured as the peak-to-peak line width using X-band
ESR spectroscopy (Jeol, Akishima, Japan).
(40.0%) of a yellow product.
1
4
– 1
[
N, D] IR (cm ): 3208–3427 (NH), 1672 (C=O), Calculated for
C
9 2 15 2 2
H D N O : C, 53.91; H, 8.55; N, 13.98. Found: C, 55.67; H, 8.20;
1
5
– 1
N, 14.40. [ N, H] IR (cm ): 3207–3428 (NH), 1678 (C=O), Calcu-
lated for C H17N NO : C, 57.99; H, 9.20; N, 15.04. Found: C,
9 2
5
(
1
5
1
5
– 1
8.06; H, 9.17; N, 15.14. [ N, D] IR (cm ): 3201-3407 (NH), 1679
1
5
C=O), Calculated for C
9
H
2
D
15
N
2
O
2
: C, 53.64; H, 8.50; N, 13.91.
Found: C, 55.18; H, 8.81; N, 14.30.
Phantom experiment
2
mM nitroxyl probes were contained in phantom tubes of vari-
3
-Methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl
ous sizes. The inner diameters of these eight tubes were 7, 6, 5, 4,
, 2, 1.5, and 0.7 mm. Typical scan conditions in OMRI were: rep-
etition time (T )/echo time (T )/ESR irradiation time (TESR):
,200 ms/25 ms/600 ms; no. of averages = 1; 64 phase-encoding
steps. The image field of view, 48 mm, was represented by a
4664 matrix.
3
(MC-PROXYL)
R
E
Concentrated sulfuric acid (97%, 0.082 mL) was added dropwise
at 58C to a stirred solution of 3-carboxy-2,2,5,5-tetramethylpyrro-
lidine-1-oxyl (2.67 mmol) in methanol (2 mL). The reaction mix-
ture was refluxed at 608C for 2 h. After evaporation, ice water
1
6
(10 mL) was added to the solution, which was then extracted
with ether. The ether layer was dried with anhydrous magne-
sium sulfate. After removal of the solvent in a rotating evapora-
tor, the residue was purified with silica gel column chromatog-
raphy to give 1.45 mmol (54.4%).
References
1
4
– 1
[
N, D] IR (cm ): 1739 (C=O), 1201 (COO), Calculated for
: C, 55.72; H, 8.42; N, 6.50. Found: C, 56.67; H, 8.76;
[1] K. Yamada, T. Nakamura, H. Utsumi, Free Radic. Res. 2006,
C
10
H
3
D
15NO
3
1
5
– 1
40, 455–460.
N, 6.59. [ N, D] IR (cm ): 1739 (C=O), 1200 (COO), Calculated for
10
1
5
C
H
18 NO
3
: C, 59.98; H, 9.06; N, 6.96. Found: C, 59.57; H, 9.08; N,
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1
5
– 1
6.97. [ N, D] IR (cm ): 1739 (C=O), 1200 (COO), Calculated for
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