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

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 322–327

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Evaluation of glucose-linked nitroxide radicals for use as an in vivo spin-label probe Shingo Sato a,⇑, Masaki Yamaguchi a, Akio Nagai a, Ryo Onuma b, Misaki Saito b, Rina Sugawara b, Sayaka Shinohara b, Eriko Okabe b, Tomohiro Ito a, Tateaki Ogata a a b

Graduate School of Science and Engineering, Yamagata University, Jonan 4-3-16, Yonezawa-shi, Yamagata 992-8510, Japan Department of Chemistry and Chemical Sciences, Faculty of Engineering, Yamagata University, Jonan 4-3-16, Yonezawa-shi, Yamagata 992-8510, Japan

g r a p h i c a l a b s t r a c t

 ESR observation of nitroxide radicals

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).

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 DPROradical.

ESR signal intensity [-/g]

h i g h l i g h t s

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Article history: Received 4 October 2013 Received in revised form 27 December 2013 Accepted 8 January 2014 Available online 21 January 2014 Keywords: Glucose-linked nitroxide Ascorbic acid Reduction ESR spectrometry White radish sprout Incorporation

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a b s t r a c t 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. Ó 2014 Elsevier B.V. All rights reserved.

Introduction

⇑ Corresponding author. Tel.: +81 238263120. E-mail address: [email protected] (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

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

S. Sato et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 322–327

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 reductants 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-Tetramethylpyrrolidine-1-oxyl (PROXYL) and 2,2,6,6-tetramethylpiperidine-1oxyl (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-hydroxy-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)-D-glucose (4), 3-hydroxymethyl-DPRO O-b-D-glucoside (5), and 6-O-(DPRO3-carbonyl)-D-glucose (6), for use as spin-label probes. New probes 4 and 6, in which the anomeric hydroxyl of glucose is free, are expected to be faster and better incorporated into plants by comparison 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 compared 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 diastereomer 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 invasive 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. 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 condensation with D-glucose was impossible, after protection of the anomeric hydroxyl of glucose by a protecting group that is possible

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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 regioselective condensation between a primary alcohol at the 6-position of the glucose and 1 or 2 was examined, followed by oxidative deprotection using cerium (IV) diammonium nitrate (CAN), which afforded the desired 4 or 6. Protection at the anomeric hydroxyl employing p-methoxyphenol 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 dicyclohexylcarbodiimide (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 saturated 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 chromatography using chloroform–methanol to afford either the desired 4 or 6 in a yield of either 57.5% or 51.0%, respectively, which was

2 2

Fig. 1. TEMPO- and DPRO-radicals, and their glucose-linked spin-label probes.

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HO HO HO

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OC HO HO

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R 7 TEMPO-4-yl 8 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-hydroxymethylDPRO (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 mT Fig. 3. ESR spectrum of 10 lM of an aqueous solution of 4 (the peak of both sides is the signal of a Mn2+ marker).

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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 timedependent 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 secondordered 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 intensity 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].

time [min] Fig. 4. Time course of reduction after the addition of 500 lM of ascorbic acid in PBS (pH 7.0) at rt (293–295 K) to each 20 lM of nitroxide aqueous solution in a 1:1 (v/v) ratio (Plots are the mean value and standard deviation, n = 3).

Table 1 Second-order rate constants, k (M1s1), 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 (M1s1)

R2

1 2 3 4 5 6

220 ± 40 22 ± 2 460 ± 40 520 ± 40 90 ± 8 148 ± 6

0.999 0.973 0.992 0.985 0.984 0.977

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) 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,

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 500 lM aqueous solution of ascorbic acid was added in a ratio of

Fig. 5. Two leaves of a white radish sprout cutting used to measure ESR spectrometry.

S. Sato et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124 (2014) 322–327 60

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time [min] 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).

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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  10 min) to give a supernatant. The resultant supernatant was rapidly measured by ESR spectrometry. Twenty ll of a 50 mM potassium ferricyanide aqueous solution was added to 100 ll 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.

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Fig. 7 shows all quantities (as shown by ESR intensity) of the radicals incorporated into the sprouts by the time point of its oxidization, 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 satisfactory for observation at each time point. Although all TEMPOradicals 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. Next, we examined by in vitro experiment what percentage of the resultant reductant was restored by ferricyanide. Reduction was conducted by the addition of 100 ll of 500 lM of an ascorbic acid solution to each 100 ll of a 20 lM 1 or 2 solution similar to the above in vitro experiment. After 30 min when reduction was almost finished, 20 ll of 50 mM potassium ferricyanide was added to 100 ll of the reaction mixture and re-oxidation was conducted. The time-course of this reaction was monitored by ESR spectrometry every 2–5 min (see S13 and 14). TEMPO-radical 1 was almost reduced by excess ascorbic acid, and most of the formed hydroxylamine was restored to the radical by the excess potassium ferricyanide, 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. The rate with respect to the sum of each nitroxide and the corresponding hydroxylamine presented in radish sprouts at each time point in Fig. 7, though the scatter was large, the rate: k (s1) 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 results 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 reactivity 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.

Conclusion Newly designed nitroxide radicals 4 and 6, which are esterlinked 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 comparison 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 reduced only by a 250-fold excess of ascorbic acid, most of them were detected without a reduction of the nitroxide radicals in radish 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 glycosides 3 and 5, respectively. This result suggests that the conjugation of the nitroxide radical and D-glucose improved the ability of a nitroxide radical to incorporate into a plant, which was further

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changed by the differences in the bonding site and form. Finally, among the 6 nitroxide samples used in this study, the DPRO-radicals 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. Experimental General The solvents used in these reactions were purified by distillation. 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. Column chromatography was performed on SephadexÒ LH-20 gel (Amersham Pharmacia Biotech AB), and flash column chromatography 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 electron 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 spectrometer. Samples were drawn into quartz capillaries (1.5  110 mm, TERUMO Co. Ltd.), the bottoms of the capillaries were sealed with putty (TERUMO Co. Ltd.) and the capillaries were placed in standard 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. Synthesis of 7 and 8 To a solution of p-methoxyphenol O-glycoside (100 mg, 0.350 mmol) and 4-carboxy-TEMPO (70 mg, 0.350 mmol) or 3-carboxy-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 reaction 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. Synthesis of 4 and 6 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 saturated NaHCO3 aqueous solution and the resultant precipitates were filtered and washed with MeOH. After the removal of organic solvents, the residue was dissolved with MeOH and separated from substances derived from CAN by SephadexÒ LH-20 gel column

chromatography (/ 2.4  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 chromatography (/ 2.0  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 Per-O-benzoyl-a-D-glucopyranosyl bromide (2209 mg, 3.35 mmol) and 3-hydroxymethyl-DPRO (380 mg, 2.23 mmol) were dissolved in dried CH2Cl2 (3 ml). To the mixture molecular sieves (MA) 4 Å powder (1.70 g) was added. To the stirred mixture silver trifluoromethanesulfonate (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 mixture, Dowex 50 W  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. 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 (cm1) 3502, 2970, 2931, 2854, 1728, 1635, 1512, 1458, 1311, 1226, 1072, 1034. 1H NMR (DMSO-d6 + D2O + phenylhydrazine) d: (TEMPO moiety) 1.00 (s, 6H, CH3  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 moiety) 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  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. ½a27 D 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) Pale-yellow prism (from AcOEt). Mp = 174–175 °C. IR m (cm1) 3469, 3369, 2970, 2978, 2931, 2837, 1711, 1635, 1508, 1460, 1354, 1290, 1219, 1084. 1H NMR (DMSO-d6 + D2O + phenylhydrazine) d: (DPRO moiety) 1.13, 1.15, 1.19 and 1.20 (each s, 3H, CH3  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  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; + H, 6.76; N, 3.03. ½a25 D 51.2 (c 1.04, CHCl3). FAB-MS (m/z) 452 (M) . 6-O-(20 ,20 ,60 ,60 -tetramethylpiperidine-10 -oxyl-40 -carbonyl)-D-glucose (4) Pale-brown powder. IR m (cm1) 3440, 2977, 2923, 1736, 1635, 1458, 1365, 1311, 1172, 1057. 13C NMR (in CD3OD + phenylhydrazine) d: (TEMPO moiety) 20.06 and 32.22 (CH4  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 C16H28NO80.5H2O: C, 51.74; H, 7.87; N, 3.77. Found: C, 51.40; H, 7.61; + N, 3.74. ½a26 D +48.4 (c 1.01, MeOH). FAB-MS (m/z) 363 (M+H) .

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HRMS (ESI+) Calcd. for C16H28NNaO8 385.17126, found 385.17181. ESR data (10 lM aqueous solution, see Fig. 4): g = 2.0057 (1: 2.0057, 3: 2.0056). aN = 1.71 mT (1: 1.71, 3: 1.70). 6-O-(20 ,20 ,50 ,50 -tetramethylpyrroline-10 -oxyl-30 -carbonyl)-D-glucose (6) Pale-brown powder. IR m (cm1) 3435, 2979, 2931, 1718, 1655, 1508, 1458, 1451, 1061. 13C NMR (in CD3OD + phenylhydrazine) d: (DPRO moiety) 25.08 (CH4  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 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 C15H24NO80.5H2O: C, 50.70; H, 7.09; N, 3.94. Found: C, 50.69; H, 7.09; N, 3.82. ½a21 D +50.1 (c 1.01, MeOH). FAB-MS (m/z) 347 (M+H)+. g = 2.0054. aN = 1.60 mT. (20 ,20 ,50 ,50 -Tetramethylpyrroline-10 -oxyl-30 -methyl) O-b-D-glucoside (5) Pale-yellow prisms (from MeOH). Mp = 153 °C. IR m (cm1) 3434, 3338, 2977, 2889, 1466, 1361, 1300, 1167, 1101, 1072, 1043. 1H NMR (in CD3OD + phenylhydrazine) d 4.27 (1H, m, H50 ), 4.45 (1H, m, H60 a), 4.66 (1H, d, J 13.0, H60 b), 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 C15H26NO7: C, 54.19; H, 7.88; N, 4.23. Found: C, 53.85; H, 8.08; N, 4.01. ½a21 42.5 (c 1.04, MeOH). FAB-MS (m/z) 332 (M)+. D g = 2.0055. aN = 1.60 mT. (3: g = 2.0055 []. aN = 1.62 mT). Analysis by ESR spectrometry 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)’. 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 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)’. Acknowledgement The authors are grateful to Professor Bunpei Hatano for his measurement of HRMS.

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Appendix A. Supplementary material 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. References [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. [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. [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. [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. [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 spinlabels, 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. [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 BF3OEt2 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. [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.