Synthesis and properties of umbelliferone-nitroxide radical hybrid compounds as fluorescence and spin-label probes

Article abstract of DOI:10.1016/j.saa.2007.09.015

Two hybrid compounds 6 and 7, linked via an ester-bond between the 7-hydroxyl residue of an umbelliferone 1 and a carboxylic acid residue of two nitroxide radicals 3 and 4, and one hybrid compound 8, linked via an ester-bond between a 3-carboxylic acid residue of umbelliferone 2 and a hydroxyl residue of nitroxide radical 5, were synthesized in good yields, and their fluorescence and ESR spectra before and after the addition of l-ascorbic acid sodium salt in PBS (pH 7.0) were measured. The ESR intensities of 6 and 7 were proportionally reduced after the addition of ascorbic acid sodium salt, and their fluorescence intensities were increased maximally by eight- and nine-fold, respectively. However, the fluorescence intensity of 8 was essentially unchanged.

Full text of DOI:10.1016/j.saa.2007.09.015

Available online at www.sciencedirect.com
Spectrochimica Acta Part A 70 (2008) 799–804
Synthesis and properties of umbelliferone-nitroxide radical
hybrid compounds as fluorescence and spin-label probes
Shingo Sato∗, Minoru Suzuki, Tomoki Soma, Minoru Tsunoda
Department of Chemistry and Chemical Engineering, Faculty of Engineering, Yamagata University,
4-3-16 Jonan, Yonezawa-shi, Yamagata 992-8510, Japan
Received 21 May 2007; received in revised form 30 August 2007; accepted 18 September 2007
Abstract
Two hybrid compounds 6 and 7, linked via an ester-bond between the 7-hydroxyl residue of an umbelliferone 1 and a carboxylic acid residue
of two nitroxide radicals 3 and 4, and one hybrid compound 8, linked via an ester-bond between a 3-carboxylic acid residue of umbelliferone
2 and a hydroxyl residue of nitroxide radical 5, were synthesized in good yields, and their fluorescence and ESR spectra before and after the
addition of l-ascorbic acid sodium salt in PBS (pH 7.0) were measured. The ESR intensities of 6 and 7 were proportionally reduced after the
addition of ascorbic acid sodium salt, and their fluorescence intensities were increased maximally by eight- and nine-fold, respectively. However,
the fluorescence intensity of 8 was essentially unchanged.
© 2007 Elsevier B.V. All rights reserved.
Keywords: Umbelliferone; Nitroxide radical; Hybrid compound; l-Ascorbic acid; ESR; Fluorescence
1. Introduction
after reduction of the nitroxide radical to a hydroxylamine
(Eq. (1)) in an aqueous solution has been previously observed
under acidic conditions [1,3,11] or in organic solvents such as
methanol, acetnitrile, or hexane [5–8,12,13], or buffer solution
[2,4,14]. However, representative fluorescent dyes such as,
fluorescein, umbelliferone, and polymethine cyanine have not
been employed as a fluorphore of these hybrid compounds.
We have been interested in the decay and enhancement of
fluorescence intensity through an electron exchange mechanism
between fluorophore and nitroxide radical in a molecule.
Fluorophore-nitroxide radical hybrid compounds have
been developed mainly for the detection of radicals or to
probe the redox reactions in biological and chemical systems,
which were applied to the detection of hydroxyl [1] and
nitric oxide [2] radicals, Fe2+ [3], and ascorbic acid [4].
In both the radical trapping and fluorescence quenching
properties of the nitroxide radical could be turned to advan-
tage in a technique for monitoring radicals and reducing
species. The fluorescence from such molecules was almost
fully quenched, presumably through an electron exchange
mechanism, which both decreased the fluorescence quantum
yield and shortened the fluorescence lifetime [5]. All of these
fluorophore-nitroxide radical hybrid compounds were linked
via an ester-bond between a naphthalene [5–9], pyrene [3] or
anthracene carboxylic acid [1,10], and a 4-hydroxy-2,2,6,6-
(1)
We here wish to report our first observation of an increase in fluo-
rescence intensity and a decrease in ESR intensity along with the
chemical reduction of the synthesized umbelliferone-nitroxide
hybrid compounds by l-ascorbic acid sodium salt in PBS (pH
7.0) [4]. The usable of PBS as a solvent is important from the
view point of applying. In these hybrid compounds, although
TEMPO-4-yl coumarin-3-carboxylate has been synthesized and
used to study the free radical kinetics of radical polymerization
[15], measurements related to the increase in fluorescence with
reduction of the nitroxide radical have not been reported.
tetramethylpiperidine-1-oxyl
3-hydroxymethyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl
(3-hydroxymethylPROXYL). The appearance of fluorescence
(4-hydroxyTEMPO)
or
∗
Corresponding author. Tel.: +81 238 263121; fax: +81 238 263121.
E-mail address: shingo-s@yz.yamagata-u.ac.jp (S. Sato).
1386-1425/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2007.09.015

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S. Sato et al. / Spectrochimica Acta Part A 70 (2008) 799–804
Scheme 1. Synthesis of umbelliferyl nitroxyl radicals (6,7, and 8). Reagents and conditions: (a) DCC (1.2 equiv), DMAP (0.1 equiv) in CH3CN, at 0 ◦C to r.t. for
7 h, Y: 72%; (b) DCC (1.0 equiv), DMAP (0.1 equiv) in CH2Cl2, at 0 ◦C to r.t. for 18 h, Y: 84%; (c) DCC (1.2 equiv), DMAP (0.3 equiv) in CH3CN, at r.t. for 18 h,
Y: 83%.
2. Results and discussion
ried out by the condensation of a 7-hydroxyl group of 1 and each
carboxylic acid of nitroxide 3 or 4, and a 3-caboxylic acid of 2
and a hydroxyl group of nitroxide 5, using DCC and DMAP in
anhydrous CH2Cl2 or CH3CN to afford hybrid compounds 6, 7
and 8 in good yield of 72, 84 and 83%, respectively.
The UV–vis and fluorescence spectral data of 1 and 2, and
the synthesized hybrid compounds 6, 7 and 8 are summarized
in Table 1. UV–vis absorption spectra of 6 and 7 were shifted
to lower wavelengths at 308 and 310 nm and their maximum
absorbance both were observed at 278 and 279 nm. Fluorescence
was observed at a very lower intensity at 450 (relative intensity:
3) and 443 (1) nm, compared with 1 [450 nm (53)], as expected.
Since the fluorescence intensity of 2 was low (0.28), and that of
its hybrid compound 8 was also low (1), it can be concluded that
hybrid compound 8 cannot be applied as a fluorescence probe
for the measurement of radicals and reducing species. Thus the
We selected two umbelliferones [7-hydroxycoumarin and
7-hydroxycoumarin-3-carboxylic acid (1 and 2)] as a flu-
orophore. Umbelliferone 1 absorbs strongly at 300 (log ε
3.9), 305 (3.95), and 325 nm (4.15), respectively, and flu-
oresces blue (λem = 455 nm) in ultraviolet and visible light,
and its fluorescence disappears in acidic media (pKa = 7.7)
[16]. Umbelliferone 1 is a representative fluorophore-biosensor
and has enjoyed extensive use in the past. We also selected
3-carboxy-2,2,5,5-tetramethylpyrroline-1-oxyl (3), 4-carboxy-
2,2,6,6-tetramethylpiperidine-1-oxyl (4-carboxyTEMPO, 4),
and 3-hydroxymethyl-2,2,5,5-tetramethylpyrroline-1-oxyl (5)
as nitroxide radicals, which are stable and are used frequently
as a spin-probe for monitoring radical and redox reactions in
biosystems [17]. The synthesis, as shown in Scheme 1, was car-
Table 1
UV–vis absorption and fluorescence spectra of umbelliferones (1, 2) and the corresponding hybrid compounds (6, 7 and 8)
Compound
Absorption in MeOH
Fluorescence (c = 1.0 ␮M) in PBS (pH 7.0)
λmax (nm)
log ε
λex (nm)
λem (nm)
Relative intensity (−)
1
6
323
278
308
279
310
292
292
4.19
4.10
4.01
4.13
3.97
4.17
4.20
325
327
450
450
53
3
7
330
443
1
2
8
307
306
408
422
0.28
1

S. Sato et al. / Spectrochimica Acta Part A 70 (2008) 799–804
801
Table 2
ESR signal intensities and g-values of the nitroxide compounds (3, 4, 5, 6, 7, 8;
10 ␮M) in 0.1 M PBS (pH 7.0)
Compound
Signal intensity
g
3
4
5
6
7
8
1.8294
1.6620
1.2210
1.5510
1.6463
0.9308
2.0058
2.0052
2.0050
2.0050
2.0052
2.0050
Fig. 3. Time-dependent ESR signal intensity after the addition of various con-
centration of l-ascorbic acid sodium salt (2500, 1000, 750, 500, 100, 50, 25 ␮M)
in 0.1 M PBS to 20 ␮M 7 in 0.1 M PBS (volume ratio 1:1).
Fig. 1. Time-resolved ESR signal intensity after the addition of 1 mL of 4 mM
l-ascorbic acid sodium salt in 0.1 M PBS to 1 mL of 20 ␮M 3 (ꢀ) or 6 (♦) in
0.1 M PBS.
fluorescence of these umbelliferone-nitroxide hybrid com-
pounds (6, 7) was scarcely observed with fluorescence
quenching due to nitoxide radicals.
The ESR intensity of 6 (1.55) was observed to be slightly
lower than that of 3 (1.83), but the ESR intensity of 7 (1.65)
was nearly the same as that of 4 (1.66), as shown in Table 2.
Furthermore, the decreasing rate of ESR intensity of 6 and 7
by the addition of l-ascorbic acid sodium salt in 0.1 M PBS
(4 mM and 500 ␮M) was also the same as that of 3 and 4,
respectively, as shown in Figs. 1 and 2. Since the difference
in the ESR intensities of 4 and 7 before and after the addition
of ascorbic acid was greater than that of 3 and 6, the rate of
reduction of both ESR intensities was equal, and the concentra-
Fig. 4. (a) Fluorescence intensity of 6 after the addition of ascorbic acid sodium
salt solution (A: 0 ␮M, B: 6 mM). (b) Fluorescence intensity of 7 after the
addition of l-ascorbic acid sodium salt solution (A: 0 ␮M, B: 6 mM). (c) Fluo-
rescence intensity of 8 after the addition of l-ascorbic acid sodium salt solution
(A: 0 ␮M, B: 6 mM).
Fig. 2. Time-resolved ESR signal intensity after the addition of 1 mL of 500 ␮M
l-ascorbic acid sodium salt in 0.1 M PBS to 20 ␮M 4 (ꢀ) or 7 (᭹) in 0.1 M PBS.

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S. Sato et al. / Spectrochimica Acta Part A 70 (2008) 799–804
Table 3
3. Conclusions
Maximum fluorescence intensity of hybrid compounds (6, 7 and 8) after the
addition of 6 mM ascorbic acid sodium salt in PBS (pH 7.0) in Fig. 5a–c
As expected, fluorescence of the synthesized hybrid com-
pounds 6, 7 and 8 was scarcely observed. By the addition of
ascorbic acid sodium salt in 0.1 M PBS (pH 7.0), the ESR
intensities of 6 and 7 were smoothly reduced, and their fluo-
rescence intensities were increased concentration dependently
in parallel. In the reduction of hybrid compounds 6, 7 and 8
by l-ascorbic acid sodium salt, the ratio of the enhancement
in fluorescence intensity for 7 was 9.08-fold, greater in com-
parison to that of 6 (8.25) and 8 (1.66) (Table 3). The ESR
sensitivity of 7 for l-ascorbic acid sodium salt was also bet-
ter than that of 6 (Figs. 1 and 2). Since umbelliferone-nitroxide
hybrid compounds 6 and 7 have structures that are different
from other hybrid compounds developed to date which are
linked between a nitroxide radical and an aromatic compound
condensed with benzene-rings such as naphthalene and pyrene
derivatives and their emission spectra were observed at longer
wavelength (443 and 450 nm), they show the potential for appli-
cation as a fluoresce spin-probe for measuring radicals such as
hydroxyl radicals in biosystems, Fe(II) in solution and ascorbic
acid in food products.
Compound
6
7
8
Relative intensitya
8.25
9.08
1.66
a
Fluorescence intensity of each hybrid compound in PBS (pH 7.0) was to
be 1.
Fig. 5. ESR signal intensity of 7 (20 ␮M) after the addition of various concen-
trations of l-ascorbic acid sodium salt.
The fluorescence intensity of the synthetic probes (6,
7) increased in proportion with the decrease in their ESR
signal intensity, indicating the conversion of the paramag-
netic nitroxide to a diamagnetic hydroxylamine. This linear
relationship between the increase in fluorescence inten-
sity and the decrease in ESR signal intensity indicates
that the change in fluorescence intensity can serve as a
sensitive means for optically detecting radicals or reduc-
ing species, such as 4-(1-naphthoyloxy)-TEMPO [8] or
4-(pyrene-1-carboxy)TEMPO [3] or 1-imino nitroxide pyrene
[11].
tion of added ascorbic acid to 4 and 7 (500 ␮M) was one-eighth
over that of 3 and 6 (4 mM), it appears that 7 would be superior
to 6 as a radical-spin probe. When an l-ascorbic acid sodium
salt solution, at a concentration over 500 ␮M, was added to a
20 ␮M PBS of 7 in a volume ratio of 1:1, the radical almost
disappeared within 20 min (see Fig. 3). Next, before and after
the addition of 6 mM l-ascorbic acid sodium salt in PBS to a
20 ␮M solution of 6, 7, or 8, the fluorescence intensity of each
compound was measured (Fig. 4a–c). The difference in the flu-
orescence intensity after the addition of l-ascorbic acid sodium
salt solution (6 mM) was 9.08-fold for 7, while that for 6 and
8 was 8.25- and 1.66-fold, respectively (see Table 3). Further-
more, while the fluorescence intensity of 7 was increased in a
concentration-dependent manner, in parallel, the ESR intensity
of 7 was decreased in a concentration-dependent manner, as
shown in Figs. 5 and 6.
4. Experimental
4.1. Apparatus
Fluorescence spectra and relative fluorescence intensity were
measured with a Hitachi 650-10M fluorescence spectropho-
tometer. The excitation and emission wavelength band passes
were both set at 2 nm. Absorption spectra were obtained on a
Hitachi U-2010 UV–vis spectrometer. Electron spin resonance
spectra were obtained on a JEOL JES-FR30 ESR spectrom-
eter. Samples were drawn into quartz capillaries, which were
then sealed at the bottom and placed standard 2 mM i.d. quartz
ESR tubes. The ESR spectrometer settings were as follows:
microwave power, 4.0 mW; frequency, 9.2 GHz; modulation
amplitude, 1.25 G. All pH values were measured with a Horiba
M-13 PH meter.
Analogous results were also observed for 6. In the case of
Fe(II) in an aqueous solution in place of ascorbic acid in PBS as
a reducing agent, the same result was also observed, though the
sensitivity was inferior.
4.2. Synthesis
The solvents used in this study 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% ethano-
lic solution of phosphomolybdic acid with heat as coloration
Fig. 6. Fluorescence intensity of 7 (20 ␮M) after the addition of various con-
centrations of l-ascorbic acid sodium salt solution.

S. Sato et al. / Spectrochimica Acta Part A 70 (2008) 799–804
803
agents. For separation and purification, flash column chromatog-
raphy was performed on silica gel (230–400 mesh, Fuji-Silysia
Co., Ltd., BW-300). Melting points were determined on a
Shibayama micro-melting point apparatus and are uncorrected.
IR spectra were recorded on a Horiba FT-720 IR spectrom-
eter using a KBr disk. NMR spectra were recorded on a
Varian Inova 500 spectrometer using Me4Si as the inter-
nal 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. Elemen-
tal analyses were performed on a Perkin–Elmer PE 2400 II
instrument.
H-3), 7.04 (dd, 1H, J 2.2, 8.4 Hz, H-6), 7.10 (d, 1H, J 2.2 Hz, H-
8), 7.50 (d, 1H, J 8.4 Hz, H-5), 7.70 (d, 1H, J 9.6 Hz, H-4). 13C
NMR (CDCl3 + hydrazobenzene) δ 19.3 (CH3 × 4), 32.2 (C2ꢀ,
6ꢀ), 41.3 (C3ꢀ, 5ꢀ), 58.3 (C4ꢀ), 110.3, 116.0, 116.6, 119.8, 128.5,
142.8 (C4), 153.2 (C8), 154.6 (C9), 160.3 (C-2), 173.2 (car-
boxy). FAB-MS (m/z) 345 (M+H)+. Anal calcd. for C19H22NO5:
C, 66.26; H, 6.44; N, 4.07. Found: C, 66.34; H, 6.37;
N, 4.06.
4.2.3. 3-(7-Hydroxycoumarinyl-4-carboxymethyl)-2,2,5,5-
tetramethylpyrroline-1-oxyl (8)
Compound 8 was synthesized by condensation of 7-
hydroxycoumarinyl-4-acetic acid (2) with 3-hydroxylmethyl-
2,2,5,5-tetramethylpyrroline-1-oxyl (5) following in the same
procedure as was used for the synthesis of 6.
Nitroxide radicals (3, 4, and 5) were synthesized by usual
manner [18,19].
Data for 8: Yellow powder. Mp 110–112 ◦C. IR ν (KBr) 1749,
4.2.1. 4-(7-O-Coumarinyl)-oxycarbonyl-2,2,6,6-
tetramethylpyrroline-1-oxyl (6)
1
1712, 1610, 1566 cm−1. H NMR (CDCl3 + hydrazobenzene)
To a stirred solution of umbelliferone (3-hydroxycoumarin 1:
50 mg, 0.31 mmol) and 3-carboxy-2,2,5,5-tetramethylpyrroline-
1-oxyl (3: 68 mg, 0.40 mmol) in dry CH3CN (8 mL) in
an ice bath, DCC (76 mg, 0.37 mmol) and DMAP (3.7 mg,
0.031 mmol) were added and the mixture was stirred at room
temperature for 7 h. The resulting precipitates were filtered
through a celite pad. The filtrate was evaporated in vacuo
and then purified by silica gel column chromatography (2:1
hexane–AcOEt) to give 6 (73 mg, 72%) as a yellow powder,
which was recrystallized from MeOH.
δ 1.26 and 1.31 (s, each 6H, CH3 × 4), 4.86 (s, 2H, CH2),
6.88 (s, 1H, H-2ꢀ), 5.73 (s, 1H, olefinic H), 7.35 (m, 2H,
ArH), 7.64 (m, 2H, ArH), 8.55 (s, 1H, –CH ). 13C NMR
(CDCl3 + hydrazobenzene) δ (PROXYL moiety) 24.4 and 25.4
(each CH3 × 2), 61.2 (CH2), 68.3 and 70.3 (C2, 5), 149.1
(C3), 132.3 (C4), (coumarin moiety) 116.8, 117.8, 125.0,
129.6, and 155.2 (benzene ring), 138.6 (C4), 156.5 (C2–C O),
162.8 (C3–C O). FAB-MS (m/z) 343 (M+H)+. Anal calcd. for
C19H20NO5: C, 66.65; H, 5.89; N, 4.09. Found: C, 66.92; H,
6.12; N, 4.14.
Data for 6: Yellow needles. Mp 161–162 ◦C. IR ν (KBr)
2979, 2931, 1739, 1620, 1348, 1290, 1269, 1012 cm−1 1H
.
Acknowledgements
NMR (CDCl3 + hydrazobenzene) δ 1.31 and 1.46 (s, each 6H,
CH3 × 4), 6.40 (d, 1H, J 9.5 Hz, H-3), 6.88 (s, 1H, H-2ꢀ),
7.15 (dd, 1H, J 2.0, 8.5 Hz, H-6), 7.17 (d, 1H, J 2.0 Hz, H-8),
7.51 (d, 1H, J 8.5 Hz, H-5), 7.69 (d, 1H, J 9.5 Hz, H-4). 13C
NMR (CDCl3 + hydrazobenzene) δ 24.6 and 24.8 (CH3 × 4),
68.0 (C5ꢀ), 69.6 (C2ꢀ), 110.4, 116.0, 116.6, 119.8, 128.5, 129.3,
135.5 (C4ꢀ), 142.8 (C4), 153.0 (C8), 154.6 (C9), 160.3 (C2),
161.0 (carboxy). FAB-MS (m/z) 329 (M+H)+. Anal calcd. for
C18H18NO5: C, 65.84; H, 5.53; N, 4.27. Found: C, 65.90; H,
5.28; N, 4.24.
The authors wish to thank Professor T. Ogata for the use of
the ESR spectrometer and helpful discussions regarding the ESR
study, Professor H. Mizuguchi for the use of the fluorescence
spectrophotometer, and Professor T. Izumi for the use of the
UV–vis spectrometer.
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