Evaluation of glucose-linked nitroxide radicals for use as an in vivo spin-label probe

Article abstract of DOI:10.1016/j.saa.2014.01.047

In vivo incorporation and reduction abilities of 4-carboxy-2,2,6,6- tetramethylpiperidine-1-oxyl (4-carboxy-TEMPO) (1), 3-carboxy-2,2,5,5- tetramethylpyrroline-1-oxyl (3-carboxy-dehydro-PROXYL, 3-carboxy-DPRO) (2), 4-hydroxy-TEMPO and 3-hydroxymethyl-DPRO O-β-D-glucosides (3 and 5), and newly designed forms of 6-O-(TEMPO-4-carbonyl and DPRO-3-carbonyl)-D-glucose (4 and 6) were evaluated using white radish sprouts. For each of these compounds, electron spin resonance (ESR) spectrometry was used to measure two effects: the rate of in vitro reduction via the addition of ascorbic acid; and, the rate of successful incorporation into radish sprouts for a reduction to the corresponding hydroxyl amine. DPRO-radicals 2, 5, and 6 were detected significantly more than TEMPO-radicals 1, 3, and 4 in vitro and in vivo for both experiments. Four glucose-linked nitroxide radicals were reduced faster than the glucose-non-linked ones in the in vitro experiment, but were nonetheless detected more each time in radish sprouts due to the absorbability. Glucose ester-linked radicals 4 and 6 were detected more than glycosides 3 and 5, which suggests that glucose ester-linked DPRO-radical 6 is the best for use as a spin-label probe that a plant will incorporate.

Full text of DOI:10.1016/j.saa.2014.01.047

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 322–327
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Evaluation of glucose-linked nitroxide radicals for use as an in vivo
spin-label probe
a
a
b
b
b
Shingo Sato a, , Masaki Yamaguchi , Akio Nagai , Ryo Onuma , Misaki Saito , Rina Sugawara ,
⇑
Sayaka Shinohara b, Eriko Okabe b, Tomohiro Ito a, Tateaki Ogata a
a Graduate School of Science and Engineering, Yamagata University, Jonan 4-3-16, Yonezawa-shi, Yamagata 992-8510, Japan
b Department of Chemistry and Chemical Sciences, Faculty of Engineering, Yamagata University, Jonan 4-3-16, Yonezawa-shi, Yamagata 992-8510, Japan
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
TEMPO- and DPRO-radicals, and their glucose-linked spin-label probes. Times-dependent ESR intensity
for the oxide of the homogenate–supernatant of two leaves from each of three white radish sprouts
soaked in 5 mM solutions of nitroxide radicals 1–6 (mean SD, n = 3).
ꢀ
ESR observation of nitroxide radicals
taken into plants and resistant to
reduction.
ꢀ
ꢀ
Glucose-linked radicals were
incorporatable into plants.
The most radical remained in plants
was a glucose ester-linked DPRO-
radical.
50
45
1
2
3
4
5
40
35
30
25
20
15
10
5
6
0
0
5
10 15 20 25 30 35 40 45 50
time [min]
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 October 2013
Received in revised form 27 December 2013
Accepted 8 January 2014
Available online 21 January 2014
In vivo incorporation and reduction abilities of 4-carboxy-2,2,6,6-tetramethylpiperidine-1-oxyl
(4-carboxy-TEMPO) (1), 3-carboxy-2,2,5,5-tetramethylpyrroline-1-oxyl (3-carboxy-dehydro-PROXYL,
3-carboxy-DPRO) (2), 4-hydroxy-TEMPO and 3-hydroxymethyl-DPRO O-b-
D-glucosides (3 and 5), and
newly designed forms of 6-O-(TEMPO-4-carbonyl and DPRO-3-carbonyl)-
D
-glucose (4 and 6) were
evaluated using white radish sprouts. For each of these compounds, electron spin resonance (ESR)
spectrometry was used to measure two effects: the rate of in vitro reduction via the addition of ascorbic
acid; and, the rate of successful incorporation into radish sprouts for a reduction to the corresponding
hydroxyl amine. DPRO-radicals 2, 5, and 6 were detected significantly more than TEMPO-radicals 1, 3,
and 4 in vitro and in vivo for both experiments. Four glucose-linked nitroxide radicals were reduced
faster than the glucose-non-linked ones in the in vitro experiment, but were nonetheless detected more
each time in radish sprouts due to the absorbability. Glucose ester-linked radicals 4 and 6 were detected
more than glycosides 3 and 5, which suggests that glucose ester-linked DPRO-radical 6 is the best for use
as a spin-label probe that a plant will incorporate.
Keywords:
Glucose-linked nitroxide
Ascorbic acid
Reduction
ESR spectrometry
White radish sprout
Incorporation
Ó 2014 Elsevier B.V. All rights reserved.
Introduction
The redox system in plants [1–5] that controls a reactive oxygen
species (ROS) that is produced in vivo has recently been observed
by ESR spectrometry [6–8]. Analysis of radicals and redox
⇑
Corresponding author. Tel.: +81 238263120.
E-mail address: shingo-s@yz.yamagata-u.ac.jp (S. Sato).
1386-1425/$ - see front matter Ó 2014 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.saa.2014.01.047

S. Sato et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 322–327
323
substances using ESR spectrometry requires a spin-label probe.
Nitroxide radicals are redox-sensitive reporter molecules, which
lose their paramagnetic moiety mainly by reactions with reduc-
tants such as ascorbic acid in plants. In vivo ESR spectrometry
using nitroxide radicals provides a noninvasive method to measure
the presence of reactive reductants through their effects on the
concentration of the nitroxide radicals [6–8]. 2,2,5,5-Tetramethyl-
pyrrolidine-1-oxyl (PROXYL) and 2,2,6,6-tetramethylpiperidine-1-
oxyl (TEMPO) are well-known and popular nitroxide-radical
spin-label probes [9–13], but their degree of incorporation into
plants is poor [7]. One of the water-soluble spin-labels is 4-hydro-
xy-TEMPO O-glucoside (4) [14–19], which is very soluble in water
and easily transported as far as the youngest leaf of a sprig of
cotoneaster [7]. As part of the ongoing development of in vivo
spin-label probes, we designed 6-O-(TEMPO-4-carbonyl)-
(4), 3-hydroxymethyl-DPRO O-b- -glucoside (5), and 6-O-(DPRO-
3-carbonyl)- -glucose (6), for use as spin-label probes. New probes
D-glucose
D
D
4 and 6, in which the anomeric hydroxyl of glucose is free, are
expected to be faster and better incorporated into plants by com-
parison with O-glucosides 3 and 5 (Fig. 1). For this study, ESR
measurement was used to gather a portion of the data that were
then used to describe the rate of in vitro reduction of nitroxide
radicals 1–6 by L-ascorbic acid [19], as well as the time-courses
for radical quantities per weight that were incorporated into white
radish sprouts [7] (Raphanus sativus, Fig. 2), which then was com-
pared with those of TEMPO- and DPRO-radicals 1 and 2, and
glucose-linked TEMPO and DPRO 3, 4, 5 and 6. The DPRO-radical,
which was used in place of PROXYL in order to avoid the diastereo-
mer formed on the conjugation with a glucose, is more sterically
shielded around the nitroxide moiety compared with the TEMPO
radical [19].
It is well-known that plants produce various redox species
when they are stressed or wounded so that a noninvasive method
such as ESR spectrometry is best when observing the redox system
in plants [6–8]. In the present study, however, we chose an inva-
sive method by cutting the plants, homogenization, obtaining a
supernatant, and oxidization for the ESR measurement in order
to separately examine each nitroxide-radical quantity incorporated
into plants, then we reported the noteworthy results.
Fig. 2. Thirteen white radish sprouts soaked in 6 ml of a 5 mM aqueous solution of
a nitroxide radical.
to deprotect under oxidative conditions, a subsequent regioselec-
tive condensation between a primary alcohol at the 6-position of
the glucose and 1 or 2 was examined, followed by oxidative depro-
tection using cerium (IV) diammonium nitrate (CAN), which affor-
ded the desired 4 or 6.
Protection at the anomeric hydroxyl employing p-methoxyphe-
nol was achieved via the glycosylation method. Next, condensation
of p-methoxyphenol O-glucoside and 1 or 2 was conducted using
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride
(EDC) and 1-hydroxybenzotriazole hydrate (HOBt) or dicyclohexyl-
carbodiimide (DCC) and N,N-dimethylaminopyridine (DMAP) in
dried DMF, yielding either 7 or 8 in a yield of either 37.8% or
43.0%, respectively. The ester-linkage at 6-OH was confirmable
from the low-field shift of H6ab in the 1H NMR spectra of 7 and
8 (7: d = 4.02 ppm for H6a, 4.33 ppm for H6b. 8: d = 4.06 ppm for
H6a, 4.39 ppm for H6b). The subsequent de-O-glycosylation of 7
or 8 was conducted using 2.0 equiv of CAN in aqueous acetonitrile
at room temperature (rt) for 2 h. After neutralization with a satu-
rated NaHCO3 aqueous solution at the conclusion of the reaction,
the resultant mixture was separated and purified by SephadexÒ
LH-20 gel using methanol followed by silica-gel column chroma-
tography using chloroform–methanol to afford either the desired
4 or 6 in a yield of either 57.5% or 51.0%, respectively, which was
Results and discussion
Synthesis
We designed two new samples of TEMPO- and DPRO-glucose (4
and 6) for use as a water-soluble spin-label probe that could be
easily incorporated into plants. The synthesis of 4 and 6 proceeded
as follows (Scheme 1). Since the synthesis of 4 by the direct con-
densation with D-glucose was impossible, after protection of the
anomeric hydroxyl of glucose by a protecting group that is possible
2
2
Fig. 1. TEMPO- and DPRO-radicals, and their glucose-linked spin-label probes.

324
S. Sato et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 322–327
R
C
O
HO
HO
O
O
(a)
O
(b)
O
OCH3
HO
HO
HO
4 or 6
O
OCH3
OH
OH
R
7
8
TEMPO-4-yl
DPRO-3-yl
Scheme 1. Synthesis of 4 and 6. Reagents and conditions: (a) 4-hydroxy-TEMPO (1 eq.), EDC (1.2 equiv), HOBt (1.2 equiv) in DMF, at rt, for 1 d, Y: 37.8% (7), 3-hydroxymethyl-
DPRO (1 eq.), DDC (1.2 equiv), DMAP (0.12 equiv) in DMF, at rt, for 16 h, Y: 43.0%, (8), (b) CAN (4.0 equiv), in CH3CN/H2O (4:1), at rt, for 2 h, Y: 57.5% (4), 51.2% (6).
1:1 (v/v) and the mixture was stirred at rt (20–22 °C). Every
3 min up to 30 min and then every 5 min up to 45 min, the ESR
signal intensity of the reaction mixture (ratio of the lowest-field
signal of the triplet signals of the sample to a signal of the Mn2+
marker, see Fig. 3) was measured three times, and each time-
dependent relative ESR peak height of 1–6 (the mean and standard
deviation for triplicate experiments) was summarized, as shown in
Fig. 5. Each initial reduction rate was calculated from the second-
1 mT
ordered curve (the data up to 10 min for samples 1, 3, and 4, up
to 20 min for sample 2, and then up to 8 min for samples 5 and
6), as shown in Table 1. Sterically shielded DPRO-nitroxides 2, 5,
and 6 were resistant to reduction and ca. 20% of them were only
reduced up to 20 min, while ca. 90% of TEMPO-radicals 1, 3, and
4 were easily reduced. The rate of the reduction of DPRO-radicals
2 or 5 and 6 was approximately 10 or 5 times slower than that of
TEMPO-radicals 1 or 3 and 4, respectively. The rate of the reduction
of glucose-linked radicals was twice that of glucose-non-linked
radicals for TEMPO, and four or six times for DPRO. The rate of
the reduction between glucose-linked radicals 3 and 4 or 5 and 6
was similar. Thus, glucose-linked radicals were reductable by
ascorbic acid compared with the non-linked radicals. Reduction
of all radical samples was finished within 20 min after the addition
of a 25-fold excess of ascorbic acid. A slight increase in ESR inten-
sity was then observed due to the re-oxidation of the resultant
hydroxylamine, which is the reductant of nitroxide, by the radical
derived from ascorbic acid (Fig. 4) [19].
Fig. 3. ESR spectrum of 10
l
M of an aqueous solution of 4 (the peak of both sides is
the signal of a Mn2+ marker).
1.2
1
1
2
3
4
5
6
0.8
0.6
0.4
0.2
0
0
10
20
30
40
50
time [min]
Fig. 4. Time course of reduction after the addition of 500
lM of ascorbic acid in PBS
Time-dependent concentration of nitroxide radicals 1–6 in white
radish sprouts: ESR measurement of each homogenate–supernatant of
three white radish sprouts, and its corresponding oxide (Figs. 6 and 7,
S1–12)
(pH 7.0) at rt (293–295 K) to each 20 M of nitroxide aqueous solution in a 1:1 (v/v)
l
ratio (Plots are the mean value and standard deviation, n = 3).
To reduce individual differences, the experiment was conducted
using three radish sprouts each time. Each experiment was carried
out 4 times, and the mean value and standard deviation were
calculated using three forms of experiment data (see S1–12,
Table 1
Second-order rate constants, k (Mꢁ1sꢁ1), for the initial rates of reduction of nitroxides
(20 lM) with ascorbic acid (500 lM) at 293–295 K (calculated on the basis of the data
from Fig. 4).
Nitroxide no.
k (Mꢁ1sꢁ1
)
R2
1
2
3
4
5
6
220 40
0.999
0.973
0.992
0.985
0.984
0.977
22
2
460 40
520 40
90
8
148
6
very soluble in water as expected and showed the characteristic
triplet splitting signal of a nitroxide radical in ESR spectrometry
(Fig. 3).
Evaluation as a spin probe
Rate of the in vitro reduction of each nitroxide radical 1–6 after the
addition of an ascorbic acid aqueous solution (Fig. 4 and Table 1)
To each 20
lM aqueous solution of nitroxide radicals 1–6, a
Fig. 5. Two leaves of
a white radish sprout cutting used to measure ESR
500 M aqueous solution of ascorbic acid was added in a ratio of
l
spectrometry.

S. Sato et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 322–327
325
60
50
40
30
20
10
0
Fig. 7 shows all quantities (as shown by ESR intensity) of the
radicals incorporated into the sprouts by the time point of its oxi-
dization, while Fig. 6 shows the radical quantities that remained
without a reduction. The TEMPO-radicals were scarcely detected
without oxidation, while the detection of DPRO-radicals was satis-
factory for observation at each time point. Although all TEMPO-
radicals were scarcely detected in Fig. 6, the ESR detection after
oxidation shown in Fig. 7, means that all TEMPO-radicals were
incorporated into the radish sprouts and were rapidly reduced to
hydroxylamine. The quantities of 4 and 6 detected in the radish
sprouts after 45 min were significantly more than those of 3 and
5, respectively.
1
2
3
4
5
6
Next, we examined by in vitro experiment what percentage of
the resultant reductant was restored by ferricyanide. Reduction
0
5
10
15
20
25
30
35
40
45
50
time [min]
was conducted by the addition of 100
acid solution to each 100 l of a 20 M 1 or 2 solution similar to
the above in vitro experiment. After 30 min when reduction was
almost finished, 20 l of 50 mM potassium ferricyanide was added
to 100 l of the reaction mixture and re-oxidation was conducted.
ll of 500 lM of an ascorbic
l
l
Fig. 6. Time-dependent ESR intensity for the homogenate–supernatant of two
leaves from each of three white radish sprouts soaked in 5 mM solutions of
nitroxide radicals 1–6 (mean + standard deviation, n = 3).
l
l
The time-course of this reaction was monitored by ESR spectrom-
etry every 2–5 min (see S13 and 14). TEMPO-radical 1 was almost
reduced by excess ascorbic acid, and most of the formed hydroxyl-
amine was restored to the radical by the excess potassium ferricy-
anide, while 20% of DPRO-radical 2 was reduced and only half of
the reduced hydroxylamine was restored to the radical because
of the steric shielding of the nitroxide moiety.
50
45
40
35
1
The rate with respect to the sum of each nitroxide and the cor-
responding hydroxylamine presented in radish sprouts at each
30
2
25
20
15
10
5
3
4
5
6
time point in Fig. 7, though the scatter was large, the rate: k (sꢁ1
)
was as follows: 1 (0.0020), 2 (0.026), 3 (0.0070), 4 (0.010), 5
(0.034), and 6 (0.031). The rate of the six- and five-membered ring
nitroxides incorporated into radish sprouts was increased on the
order of 4 > 3 > 1 and 5 > 6 > 2, respectively.
Considering the above in vivo experiment on the basis of the re-
sults of the in vitro experiment, the quantity of the TEMPO-radicals
detected in the radish sprouts after oxidation was significantly less
than that of the DPRO-radicals. This difference was due to the reac-
tivity of the TEMPO-radicals. This suggests that the reactive TEMPO
radical was converted to the other restore-impossible diamagnetic
compounds except for the hydroxylamine in radish sprouts.
0
0
5
10
15
20
25
30
35
40
45
50
time [min]
Fig. 7. Time-dependent ESR intensity for the oxide of the homogenate–supernatant
of two leaves from each of three white radish sprouts soaked in 5 mM solutions of
nitroxide radicals 1–6 (mean + standard deviation, n = 3).
Supporting Information (SI)). To 6 ml of a 5 mM solution of each
radical in water, 13 white radish sprouts (ca. 10 cm, see Fig. 2) with
similar-sized roots were soaked at rt (at 20–22 °C). After 5, 15, 30,
and 45 min, three stalks with two leaves of each white radish
sprout were quickly cut, weighed (see Fig. 5, 0.18–0.36 g), and
homogenized with 1.5 ml of PBS (0.1 M, pH7.0), and the resultant
homogenate was centrifuged (12,000 rpm ꢂ 10 min) to give a
supernatant. The resultant supernatant was rapidly measured by
Conclusion
Newly designed nitroxide radicals 4 and 6, which are ester-
linked at the 6-position of the glucose, were synthesized and their
rate of in vitro reduction by ascorbic acid and in vivo incorporation
and reduction abilities in white radish sprouts were explored by
ESR spectrometry, and compared with that of glycoside radicals 3
and 5, and glucose-non-linked radicals 1 and 2. During the
in vitro experiment, each of the glucose-linked radicals 3 and 4,
or 5 and 6 were reduced 2–3 times or 5–7 times faster in compar-
ison with non-linked radicals 1 or 2, respectively. During the
in vivo experiment, though ESR detection of the TEMPO-radicals
was scarcely observed in the supernatants, 45 min after oxidation
it had increased on the order of 4 > 3 > 1, and 6 > 5 > 2. The low
incorporation ability of glucose-non-linked radicals 1 and 2 into
plants was as expected. Since 20% of the DPRO-radicals were re-
duced only by a 250-fold excess of ascorbic acid, most of them
were detected without a reduction of the nitroxide radicals in rad-
ish sprouts and were significantly increased compared with the
TEMPO-radicals at each time point. The ESR detection of 4 and 6
linked by ester-bonding after 45 min was more than that of glyco-
sides 3 and 5, respectively. This result suggests that the conjuga-
ESR spectrometry. Twenty
ll of a 50 mM potassium ferricyanide
aqueous solution was added to 100
l
l of the supernatant and
mixed via mixer, whereby re-oxidation of the hydroxylamine
was carried out. The oxide solution was measured each time by
ESR spectrometry (all ESR measurements of each oxide sample
was conducted after 30 min of mixing). The ESR spectra of all
samples showed the characteristic triplet signals derived from a
nitroxide radical. ESR signal intensity per weight (the ratio of a
lower-field signal of the triplet signals of a sample to the signal
of a Mn2+ marker per weight of three cut radish sprout stalks)
was calculated for each time priod (5, 15, 30, and 45 min), and
the mean value and standard deviation of the three experiments
(n = 3) are shown in Fig. 6. The time-dependent ESR signal intensity
per weight of the oxide sample was calculated considering a
1.2-fold dilution of the supernatant sample, and the mean value
and standard deviation (n = 3) are shown in Fig. 7.
tion of the nitroxide radical and
a nitroxide radical to incorporate into a plant, which was further
D-glucose improved the ability of

326
S. Sato et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 322–327
changed by the differences in the bonding site and form. Finally,
among the 6 nitroxide samples used in this study, the DPRO-radi-
cals were resistant to invasive operations such as cutting and
homogenization, and glucose-ester-linked DPRO 6 was the best
for use as an in vivo spin-label probe that a plant could incorporate.
chromatography (/ 2.4 ꢂ 80 cm) with MeOH. After the removal
of MeOH, the fractions including 4 or 6 (TLC:CHCl3–MeOH = 10:1,
Rf = 0.33) were further purified by silica-gel column chromatogra-
phy (/ 2.0 ꢂ 8.0 cm, CHCl3–MeOH = 10:1) to give 4 (148 mg, Y:
57.5%) or 6 (126 mg, 51.2%) as a pale-brown powder.
Synthesis of 5
Experimental
Per-O-benzoyl-a-D-glucopyranosyl bromide (2209 mg, 3.35 mmol)
General
and 3-hydroxymethyl-DPRO (380 mg, 2.23 mmol) were dissolved
in dried CH2Cl2 (3 ml). To the mixture molecular sieves (MA) 4Å
powder (1.70 g) was added. To the stirred mixture silver trifluoro-
methanesulfonate (AgOTf, 861 mg, 3.35 mmol) was added in an ice
bath and the resultant mixture was stirred in the dark at rt for
overnight. The reaction mixture was added into ice-cold water
and extracted twice with CHCl3. The organic layer was washed
with brine and dried with MgSO4 and solvents were evaporated
in vacuo. The residual solid was dissolved in dried THF (3 ml) and
MeOH (6 ml). To the mixture, 25% NaOMe (0.5 ml) in MeOH was
added, and it was stirred at rt for 1 h. To the stirred reaction mix-
ture, Dowex 50 W ꢂ 8 (H+) resin was added until the resultant
mixture was neutralized. After evaporation, the residual solid
was separated by silica-gel column chromatography (CHCl3–
MeOH = 10:1) to give 5 (389 mg, 52.5%) as pale-yellow prisms.
The solvents used in these reactions were purified by distilla-
tion. Reactions were monitored by TLC on 0.25 mm silica-gel
F254 plates (E. Merck) using UV light, and a 7% ethanolic solution
of phosphomolybdic acid with heat as the coloration agents. Col-
umn chromatography was performed on SephadexÒ LH-20 gel
(Amersham Pharmacia Biotech AB), and flash column chromatog-
raphy was performed on silica-gel (40–50 lm, Kanto Reagents
Co. Ltd., silica-gel 60) to separate and purify the reaction products.
Optical rotations were recorded on a JASCO DIP-370 polarimeter.
Melting points were determined using an ASONE micro-melting
point apparatus and uncorrected values are reported. IR spectra
were recorded on a Horiba FT-720 IR spectrometer using a KBr
disk. NMR spectra were recorded on a JEOL ECX-500 spectrometer
using Me4Si as the internal standard. Mass spectral data were
obtained by fast-atom bombardment (FAB) using 3-nitrobenzyl
alcohol (NBA) as a matrix on a JEOL JMS-AX505HA instrument.
High-resolution mass spectra (HRMS) were obtained under elec-
tron spray ionization (ESI) conditions on a JEOL JMS-T100LP.
Elemental analyses were performed on a Perkin–Elmer PE 2400 II
instrument. After being dried over 80 °C under reduced pressure
for more than 2 h, each product was subjected to elemental
analysis.
ESR spectra were obtained on a JEOL JES-FR30 ESR spectrome-
ter. Samples were drawn into quartz capillaries (1.5 ꢂ 110 mm,
TERUMO Co. Ltd.), the bottoms of the capillaries were sealed with
putty (TERUMO Co. Ltd.) and the capillaries were placed in stan-
dard 2 mm-i.d. quartz ESR tubes. The ESR spectrometer settings
were as follows: microwave power, 4.0 mW; frequency,
9.1896 GHz; and, modulation amplitude, 1.25 G. White radish
sprouts (Sanwa norin Co. Ltd., Japan) were purchased from a green
grocery on the morning of the day.
Data of the new compounds 4, 5, 6, 7, and 8
p-Methoxyphenyl O-{6-O-(TEMPO-4-carbonyl)}-b-
D
-glucoside (7)
Pale-yellow prism (from AcOEt). Mp = 170–172 °C. IR
m
(cmꢁ1
)
3502, 2970, 2931, 2854, 1728, 1635, 1512, 1458, 1311, 1226,
1072, 1034. 1H NMR (DMSO-d6 + D2O + phenylhydrazine) d: (TEM-
PO moiety) 1.00 (s, 6H, CH3 ꢂ 2), 1.03 and 1.05 (each s, 3H, CH3),
1.41 and 1.68 (each m, 1H, CH2), 2.62 (m, 1H > CHA), (glucose moi-
ety) 3.12 (t, 1H, J 9.1 Hz, H2), 3.22 (t, 1H, J 9.1 Hz, H3), 3.28 (t, 1H, J
9.1 Hz, H4), 3.60 (m, 1H, H5), 4.02 (dd, 1H, J 12.1, 8.0 Hz, H6a), 4.33
(dd, 1H, J 12.1, 1.9 Hz, H6b), 4.74 (d, 1H, J 7.6 Hz, H1), 5.21, 5.33,
and 5.40 (each d, 1H, J 5.3 Hz, OH ꢂ 3), (p-CH3OPh moiety) 3.66
(s, 3H, CH3OA), 6.71 (d, 2H, J 8.3 Hz, p-substituted ArH), 6.94 (d,
2H, J 8.3 Hz, p-substituted ArH). Anal. Calcd. for C23H34NO9: C,
58.96; H, 7.32; N, 2.99. Found: C, 58.65; H, 7.52; N, 3.13. ½a D27
ꢃ
ꢁ26.7 (c 1.01, CHCl3). FAB-MS (m/z) 469 (M+H)+.
p-Methoxyphenyl O-{6-O-(DPRO-3-carbonyl)}-b-
D
-glucoside (8)
(cmꢁ1
)
Synthesis of 7 and 8
Pale-yellow prism (from AcOEt). Mp = 174–175 °C. IR
m
3469, 3369, 2970, 2978, 2931, 2837, 1711, 1635, 1508, 1460,
1354, 1290, 1219, 1084. 1H NMR (DMSO-d6 + D2O + phenylhydr-
azine) d: (DPRO moiety) 1.13, 1.15, 1.19 and 1.20 (each s, 3H,
CH3 ꢂ 4), (glucose moiety) 3.14 (t, 1H, J 8.8 Hz, H2), 3.21 (t, 1H, J
8.8 Hz, H3), 3.28 (t, 1H, J 8.8 Hz, H4), 3.60 (m, 1H, H5), 4.06 (dd,
1H, J 12.2, 7.6 Hz, H6a), 4.39 (br. d, 1H, J 12.2 Hz, H6b), 5.20 (d,
1H, J 4.8 Hz, OH), 5.20, 5.33, and 5.38 (each d, 1H, J 5.4 Hz, OH
ꢂ 3), (p-CH3OPh moiety) 3.67 (s, 3H, CH3OA), 6.71 (d, 2H, J
8.1 Hz, p-substituted ArH), 7.09 (d, 2H, J 8.1 Hz, p-substituted
ArH). Anal. Calcd. for: C, 58.10; H, 6.68; N, 3.10. Found: C, 58.27;
To
a solution of p-methoxyphenol O-glycoside (100 mg,
0.350 mmol) and 4-carboxy-TEMPO (70 mg, 0.350 mmol) or 3-car-
boxy-DPRO (64 mg, 0.350 mmol) in dried DMF (0.5 ml), EDC
(74.0 mg, 0.385 mmol) and HOBt (52.0 mg, 0.385 mmol) or DCC
(587 mg, 0.42 mmol) and DMAP (5 mg, 0.042 mmol) were added
and the mixture was stirred at rt for 1 day or overnight. The reac-
tion mixture was diluted with chloroform (20 ml) and directly
purified by silica-gel column chromatography (CHCl3–
MeOH = 20:1 and 10:1) to give 8 (62.0 mg, Y: 37.8%) or 9 (68 mg,
43.0%) as a pale-red crystal.
H, 6.76; N, 3.03. ½a D25
ꢃ
ꢁ51.2 (c 1.04, CHCl3). FAB-MS (m/z) 452 (M)+.
Synthesis of 4 and 6
6-O-(20,20,60,60-tetramethylpiperidine-10-oxyl-40-carbonyl)-
D-glucose
(4)
To a stirred solution of 7 (333 mg, 0.711 mmol) or 8 (321 mg,
0.711 mmol) in CH3CN (4 ml) and H2O (1.2 ml), CAN (1560 mg,
2.85 mmol) was added and the mixture was stirred at rt for 2 h.
The reaction mixture was neutralized with the addition of a satu-
rated NaHCO3 aqueous solution and the resultant precipitates were
filtered and washed with MeOH. After the removal of organic sol-
vents, the residue was dissolved with MeOH and separated from
substances derived from CAN by SephadexÒ LH-20 gel column
Pale-brown powder. IR m
(cmꢁ1) 3440, 2977, 2923, 1736, 1635,
1458, 1365, 1311, 1172, 1057. 13C NMR (in CD3OD + phenylhydr-
azine) d: (TEMPO moiety) 20.06 and 32.22 (CH4 ꢂ 4), 35.85,
35.90, 42.27, 59.42, and 177.19 and 177.24 (C@O), (glucose moiety,
mainly a mixture of a- and b-pyranose) 64.96, 70.47, 71.38, 71.59,
73.47, 74.46, 75.02, 75.91, 77.57, 93.76, 97.89. Calcd. for C16H28-
NO8ꢄ0.5H2O: C, 51.74; H, 7.87; N, 3.77. Found: C, 51.40; H, 7.61;
N, 3.74. ½a 2D6
ꢃ
+48.4 (c 1.01, MeOH). FAB-MS (m/z) 363 (M+H)+.

S. Sato et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 322–327
327
HRMS (ESI+) Calcd. for C16H28NNaO8 385.17126, found 385.17181.
ESR data (10 M aqueous solution, see Fig. 4): g = 2.0057 (1:
Appendix A. Supplementary material
l
2.0057, 3: 2.0056). aN = 1.71 mT (1: 1.71, 3: 1.70).
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.saa.2014.01.047.
6-O-(20,20,50,50-tetramethylpyrroline-10-oxyl-30-carbonyl)-
(6)
D-glucose
References
Pale-brown powder. IR
m
(cmꢁ1) 3435, 2979, 2931, 1718, 1655,
1508, 1458, 1451, 1061. 13C NMR (in CD3OD + phenylhydrazine) d:
(DPRO moiety) 25.08 (CH4 ꢂ 4), 62.27, 62.36, 68.45, 137.40, 137.50,
148.13, and 165.16 and 165.34 (C@O), (glucose moiety, mainly a
[1] U. Herber, Monodehydroascorbate radical detected by electron paramagnetic
resonance spectrometry is a sensitive probe of oxidative stress intact leaves,
Plant Cell Physiol. 37 (1966) 1066–1072.
[2] N. Doke, Involvement of superoxide anion generation in the hypersensitive
response of potato tuber tissues to infection with an incompatible race of
Phytophthora infestans and to the hyphal wall components, Physiol. Plant
Pathol. 23 (1983) 345–357.
[3] N. Doke, Generation of superoxide anion by potato tuber protoplasts during
the hypersensitive response to hyphal wall components of Phytophthora
infestans and specific inhibition of the reaction by suppressors of
hypersensitivity, Physiol. Plant Pathol. 23 (1983) 359–367.
mixture of
a- and b-pyranose) 64.27, 64.41, 64.68, 70.17, 70.39,
71.31, 71.51, 71.57, 71.68, 72.64, 73.53, 74.32, 74.98, 75.90,
77.45, 93.74, 97.87. Anal. Calcd. for C15H24NO8ꢄ0.5H2O: C, 50.70;
H, 7.09; N, 3.94. Found: C, 50.69; H, 7.09; N, 3.82. ½a D21
ꢃ
+50.1 (c
1.01, MeOH). FAB-MS (m/z) 347 (M+H)+. g = 2.0054. aN = 1.60 mT.
[4] Y. Miura, H. Yoshioka, N. Doke, CDNA cloning of sesquiterpene cyclase and
squalene synthase, and expression of the genes in potato tuber infected with
Phytophthora infestans, Plant Sci. 105 (1995) 45–52.
[5] H. Yoshioka, F. Bouteau, T. Kawano, Discovery of oxidative burst in the field of
plant immunity, Plant Signal Behav. 3 (3) (2008) 153–155.
(20,20,50,50-Tetramethylpyrroline-10-oxyl-30-methyl) O-b-
(5)
D
-glucoside
Pale-yellow prisms (from MeOH). Mp = 153 °C. IR
m
(cmꢁ1
)
3434, 3338, 2977, 2889, 1466, 1361, 1300, 1167, 1101, 1072,
[6] M. Tada, T. Shiraishi, H. Yokoyama, H. Ohya, T. Ogata, H. Kamada,
Nondestructive real-time monitoring of the redox status in a potted plant by
using a surface-coil-type ESR resonator, Chem. Lett. (2001) 1122–1123.
[7] M. Aoyama, H. Ohya, S. Sato, T. Ogata, A drastic redox response in plants by
environmental stress observed through a novel spin probe, ITE Lett. Batteries
New Technol. Med. 5 (5) (2004) 492–495.
[8] M. Endo, H. Kurosawa, T. Kawai, T. Ito, T. Ogata, In vivo real-time detection of
plant response to physical and chemical stresses by spin probe ESR, Chem. Lett.
41 (2012) 1584–1585.
[9] A.R. Forrester, J.M. Hay, R.H. Thomson (Eds.), Organic Chemistry of Stable Free
Radicals, Academic, London, 1968.
[10] E.G. Rozantsev, in: H. Ulrich (Ed.), In Free Nitroxyl Radicals, Plenum, New York,
1970.
1
1043. H NMR (in CD3OD + phenylhydrazine) d 4.27 (1H, m, H50),
4.45 (1H, m, H60a), 4.66 (1H, d, J 13.0, H60b), 5.33 (1H, d, J 9.5,
H10), 5.74 (1H, t, J 10.5, H20), 5.78 (1H, t, J 10.5, H40), 6.26 (1H, t, J
9.5, H30). 13C NMR (CD3OD) d: (glucose moiety) 65.93, 69.13,
71.42, 74.89, 77.91, 103.36, (DPRO moiety) 24.59, 24.63, 25.54,
25.63, 62.656, 69.13, 71.4, 131.60, 142.14. Anal. Calcd. for
C
15H26NO7: C, 54.19; H, 7.88; N, 4.23. Found: C, 53.85; H, 8.08;
N, 4.01. ½a 2D1
ꢃ
ꢁ42.5 (c 1.04, MeOH). FAB-MS (m/z) 332 (M)+.
g = 2.0055. aN = 1.60 mT. (3: g = 2.0055 [ꢁ]. aN = 1.62 mT).
[11] L.J. Berliner (Ed.), Spin Labeling: Theory and Applications, Academic, New York,
1976.
[12] J.F.W. Keana, Newer aspects of the synthesis and chemistry of nitroxide spin
labels, Chem. Rev. 78 (1978) 37–64.
Analysis by ESR spectrometry
[13] L.B. Volodarsky, V.A. Reznikov, V.I. Ovcharenko (Eds.), Synthetic Chemistry of
Stable Nitroxides, CRS, Boca Raton, FL, 1994.
[14] N.R. Plessas, I.J. Goldstein, Synthesis of a- and b-glycosides containing spin-
labels, as probes for studies of carbohydrate–protein interaction, Carbohydr.
Res. 89 (1981) 211–220.
[15] T. Gnewuch, G. Sosnovsky, Spin-labeled carbohydrates, Chem. Rev. 86 (1986)
203–238.
In vitro measurement of the reduction rate of each of the nitroxide
radicals (1–6) after the addition of an ascorbic acid aqueous solution
Experiment was showed in the section ‘rate of the in vitro
reduction of each nitroxide radical 1–6 after the addition of an
ascorbic acid aqueous solution (Fig. 4 and Table 1)’.
[16] S. Sato, T. Kumazawa, S. Matsuba, J. Onodera, M. Aoyama, H. Obara, H. Kamada,
Novel glycosylation of the nitroxyl radicals with peracetylated glycosyl
fluorides using a combination of BF3ꢄOEt2 and an amine base as promoters,
Carbohydr. Res. 334 (2001) 215–222.
[17] S. Sato, M. Nemoto, T. Kumazawa, S. Matsuba, J. Onodera, M. Aoyama, H. Obara,
H. Kamada, Synthesis and enzyme-catalyzed hydrolysis of a radical-masked
glycosylated spin-label reagent, Carbohydr. Res. 339 (2004) 2425–2432.
[18] K. Fukui, T. Ito, M. Tada, M. Aoyama, S. Sato, J. Onodera, H. Ohya, Solution-state
dynamics of sugar-connected spin probes in sucrose solution as studied by
multiband (L-, X-, and W-band) electron paramagnetic resonance, J. Magn.
Reson. 163 (2003) 174–181.
In vivo measurement of the incorporation rate of the nitroxide radicals
(1–6) into white radish sprouts
Experiment was showed in the section ‘time-dependent con-
centration of nitroxide radicals 1–6 in white radish sprouts: ESR
measurement of each homogenate–supernatant of three white
radish sprouts, and its corresponding oxide (Figs. 6 and 7, S1–12)’.
Acknowledgement
[19] J.T. Paletta, M. Pink, B. Foley, S. Rajca, A. Rajca, Synthesis and reduction kinetics
of sterically shielded pyrrolidine nitroxides, Org. Lett. 14 (20) (2012) 5322–
5325.
The authors are grateful to Professor Bunpei Hatano for his
measurement of HRMS.