Synthesis and spectral properties of polymethine-cyanine dye-nitroxide radical hybrid compounds for use as fluorescence probes to monitor reducing species and radicals

Article abstract of DOI:10.1016/j.saa.2008.07.045

Various hybrid compounds comprised of two types of nitroxide radicals and either a pentamethine (Cy5) or trimethine cyanine (Cy3) were synthesized. The nitroxide radicals were linked either via an ester-bond to one or two N-alkyl carboxyl-terminated groups of Cy5, or via two amido-bonds (aminocarbonyl or carbonylamino group) to the 5-position of the indolenine moieties of Cy5 and Cy3. Changes in fluorescence and ESR intensities of the hybrid compounds were measured before and after addition of Na ascorbate in PBS (pH 7.0) to reduce the radicals. Among the hybrid compounds synthesized, those that linked the nitroxide radicals via an aminocarbonyl residue at the 5-position of the indolenine moieties on Cy5 and Cy3 exhibited a 1.8- and 5.1-fold increase in fluorescence intensity with the reduction of the nitroxide segment by the addition of Na ascorbate, respectively. In contrast, fluorescence intensity was not enhanced in the other hybrid compounds. Thus, the hybrid compounds which exhibited an increase in fluorescent intensity with radical reduction can be used in the quantitative measurement of reducing species such as Fe²⁺ and ascorbic acid, and hydroxyl radicals. Because these hybrid compounds have the advantage of fluorescing at longer wavelengths-661 (Cy5) or 568 (Cy3) nm, respectively, they can be used to measure radical-reducing species or radicals either in solution or in vivo.

Full text of DOI:10.1016/j.saa.2008.07.045

Spectrochimica Acta Part A 71 (2009) 2030–2039
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Synthesis and spectral properties of polymethine-cyanine dye–nitroxide
radical hybrid compounds for use as fluorescence probes to monitor
reducing species and radicals
Shingo Satoa,∗, Minoru Tsunodaa, Minoru Suzukia, Masahiro Kutsunaa, Kiyomi Takido-uchia,
Mitsuru Shindoa, Hitoshi Mizuguchia, Heitaro Obarab, Hiroaki Ohyab
a Graduate School of Science and Engineering, Yamagata University, 4-3-16 Jonan, Yonezawa-shi, Yamagata 992-8510, Japan
b Institute for Life Support Technology, Yamagata Promotional Organization for Industrial Technology, 2-2-1 Matsuei, Yamagata 990-2473, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Various hybrid compounds comprised of two types of nitroxide radicals and either a pentamethine (Cy5)
or trimethine cyanine (Cy3) were synthesized. The nitroxide radicals were linked either via an ester-bond
to one or two N-alkyl carboxyl-terminated groups of Cy5, or via two amido-bonds (aminocarbonyl or
carbonylamino group) to the 5-position of the indolenine moieties of Cy5 and Cy3. Changes in fluorescence
and ESR intensities of the hybrid compounds were measured before and after addition of Na ascorbate
in PBS (pH 7.0) to reduce the radicals. Among the hybrid compounds synthesized, those that linked the
nitroxide radicals via an aminocarbonyl residue at the 5-position of the indolenine moieties on Cy5 and
Cy3 exhibited a 1.8- and 5.1-fold increase in fluorescence intensity with the reduction of the nitroxide
segment by the addition of Na ascorbate, respectively. In contrast, fluorescence intensity was not enhanced
in the other hybrid compounds. Thus, the hybrid compounds which exhibited an increase in fluorescent
intensity with radical reduction can be used in the quantitative measurement of reducing species such
as Fe2+ and ascorbic acid, and hydroxyl radicals. Because these hybrid compounds have the advantage of
fluorescing at longer wavelengths—661 (Cy5) or 568 (Cy3) nm, respectively, they can be used to measure
radical-reducing species or radicals either in solution or in vivo.
Received 14 March 2008
Received in revised form 24 July 2008
Accepted 27 July 2008
Keywords:
Cy5
Cy3
Nitroxide radical
ESR intensity
Fluorescence intensity
© 2008 Elsevier B.V. All rights reserved.
1. Introduction
cence technique makes it possible to visualize the presence of the
radicals or radical-reducing species, in addition to the quantitative
Previously, hybrid compounds comprised of a nitroxide radical
and a fluorophore linked via an amido- or ester-bond as a spacer
had been prepared and their use in quantifying radical and radical-
reducing species has been evaluated (Fig. 1) [1]. Their fluorescence
was mostly faded owing to electron transfer to nitroxide radicals,
but was recovered by reducing or radical trapping of the nitroxide
radicals. A method for the quantitative measurement of reductants
such as Fe2+ [1-a], ascorbic acid [1-b], or of radical-trapping species
such as hydroxyl radicals [1-c,d,e] was developed by applying this
phenomena. The fluorescence technique allows detection of antiox-
idants in the submicromolar concentration scale while the electron
spin resonance (ESR) technique can detect only micromolar con-
centrations [1-f]. The ESR measurements are limited by the high
cost and complexity of the required equipment and by difficulties
in acquiring and processing experimental data [1-b]. The fluores-
measurement of them [1-g].
For the preparation of these hybrid compounds, 2,2,6,6-tetra-
methylpiperidine-1-oxyl (TEMPO) or 2,2,5,5,-tetramethylpyrroli-
dine-1-oxyl (PROXYL) has been used as the stable nitroxide rad-
ical, and aromatic compounds, such as naphthalene [1-b,h,i,j,d]
or pyrene [1-a,b,k,l], had been used as the fluorescence molecule.
These aromatic compounds have short excitation wavelength (ꢀex
300 and 350 nm), and the emission wavelengths (ꢀem) also are
short—390 and 430 nm. Both the excitation and emission wave-
lengths often overlap the absorption spectra of proteins and the
other background compounds [2-a,b,c,d]. It is unclear how hybrid
compounds composed of non-aromatic fluorochromes and nitrox-
ide radicals affect the recovery of fluorescence with the reduction
of radicals.
We synthesized hybrid compounds composed of well-known
fluorescent molecules and nitroxide radicals and evaluated the flu-
orescence and ESR intensities of the hybrid compounds. In a previ-
ous paper [3], three hybrid compounds composed of a well-known
fluorophore, umbelliferone, and 2,2,5,5,-tetramethylpyrroline-1-
∗
Corresponding author. Tel.: +88 238 26 3120; fax: +88 238 26 3120.
E-mail address: shingo-s@yz.yamagata-u.ac.jp (S. Sato).
1386-1425/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2008.07.045

S. Sato et al. / Spectrochimica Acta Part A 71 (2009) 2030–2039
2031
cyclic nucleus and the number of double bonds in the polymethine
chain [2-e].
2. Results and discussion
First, an ester-linkage between a hydroxyl group of a nitrox-
ide radical (Fig. 3) and the N-alkyl carboxyl-terminated group of
Cy5 was formed. The N-alkyl carboxyl-terminated group generally
functions as a spacer for protein binding [2-f]. One of two radicals,
4-hydroxy-TEMPO (4) or 3-hydroxymethyl-TEMN (5) was bonded
either to both or to one of two terminal carboxyl residues. The N-
alkyl carboxyl residue is composed of either a two- or five-carbon
chain. A total of eight hybrid compounds were synthesized (Fig. 4).
ESR and fluorescence spectra of these hybrid compounds were mea-
sured in phosphate-buffered saline (PBS, 0.1 M, pH 7.0). However,
both ESR and fluorescence intensities were much lower than an
original nitroxide radical or Cy5, and each intensity value was var-
ious. When excess of Na ascorbate was added to a solution of these
hybrid compounds, ESR intensity was decayed, however all fluo-
rescence intensities were not enhanced. Hydroxylamine acetate of
these hybrid compounds which is a diamagnetic derivative and is
not subjected to oxidation (Fig. 1) was also observed no enhance-
ment of fluorescence intensity.
Since the methyl ester of N-alkyl-terminated carboxylic acid
also did not exhibit fluorescence, the development of this series of
hybrid compounds for use as fluorescence probes in the detection
of radical reducing species was stopped. Blough and co-workers
reported that relatively high rates of quenching by nitroxide rad-
ical (>108 s−1) are observed over distances as great as 12 Å [1-i].
Unfortunately, our results differed from theirs.
We next planed a design of the hybrid compounds linked at the
closer position of the fluorophore with a nitroxide radical (Fig. 5).
Two nitroxide-equivalents were introduced at the 5-position of
the indolenine skeleton in both Cy5 and Cy3 via an amido-bond.
A methyl group was selected as the N-alkyl residue because of
unnecessary of the spacer in this study. Based on trial and error,
a successful synthesis was finally achieved by the following reac-
tion sequence, as shown in Scheme 1: (1) synthesis of methyl
5-nitroindolenium; (2) reaction with 1,1,3,3-tetraethoxypropane
and triethyl orthoformate, leading to 5-nitro-Cy5 (9) and 5-
nitro-Cy3 (15); (3) reduction of the two nitro groups to amino
groups (5-amino-Cy5 10 and -Cy3 16); and (4) condensation of
10 and 16 with 3-carboxy-2,2,5,5-tetramethylpyrroline-1-oxyl (3-
carboxy-TEMN 7, Fig. 3).
Fig. 1. Mechanism of the fluorescent increase and decrease in a fluorophore nitrox-
ide hybrid compound.
oxyl (TEMN) or TEMPO radicals (1, 2, and 3) were synthesized
and the relationships between their fluorescence and ESR inten-
sities before and after addition of l-ascorbic acid sodium salt (Na
ascorbate) were explored (Fig. 2). It appeared that the decay in ESR
intensity upon addition of Na ascorbate caused a proportional and
parallel increase in fluorescence intensity. Because fluorescence
intensity increased in a concentration-dependent manner to a max-
imal eight- or nine-fold elevation, these two umbelliferylnitroxide
radical-hybrid compounds 1 and 2 may be used as fluorescence
spin-probes for the quantitative measurement of radicals in biosys-
tems, Fe2+ in solution, and ascorbic acid in food products [1-d]. The
excitation and emission wavelengths of these compounds were 330
and 443 nm, respectively, which were a little longer than those of
pyrene-TEMPO (ꢀem 430 nm) [1-c]. However, the wavelengths are
not long enough to perform measurements in living cells and organ-
isms due to autofluorescence from normal cellular constituents
[2-a,b,c,d].
This paper introduces two new hybrid compounds composed of
nitroxide radicals and well-known polymethine-cyanine dyes (Cy3
and Cy5), which are typically used as fluorescent labels for pro-
teins, as the fluorescence molecules. Cy3 and Cy5 have the following
properties: emission wavelengths of 500 and 650 nm, respectively:
sharp and intense absorption bands (ε > 100,000): and, in ethanol,
quantum fluorescence yield is typically between 0.2 and 0.4 [2-a].
Cy3 and Cy5 are conjugated polymethine fluorophores and differ
significantly from aromatic compound such as naphthalene and
pyrene which have been used previously in the synthesis of hybrid
compounds. The advantage of using cyanine dyes is that these dyes
can be tuned to the desired wavelengths by altering the hetero-
Fig. 2. Umbelliferylnitroxide radical hybrid compounds.
Fig. 3. Nitroxide radicals adapted for the synthesis of hybrid compounds.

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S. Sato et al. / Spectrochimica Acta Part A 71 (2009) 2030–2039
Fig. 4. Cy5-nitroxide hybrid compounds linked between one or both of N-alkyl-terminated carboxylic acids and nitroxide radicals (4 and 5) via ester bond.
Fig. 5. Cy3, Cy5-nitroxide compounds linked between at the 5-positions of cyanine dyes and nitroxides via two kinds of amido bonds (a: aminocarbonyl group, b: carbony-
lamino group).
Fluorescence intensities of Cy5 (8) in some solvents, as mea-
sured, were found to vary (Fig. 6). When acetonitrile was used
as a solvent, fluorescence intensity reached maximum; there-
after acetonitrile was employed as the solvent in the fluorescence
measurement. Na ascorbate used for the reduction of the hybrid
compounds was all dissolved in PBS (0.1 M, pH 7.0). The influence
of the fluorescence intensity was explored when a PBS solution of
Na ascorbate was added to an acetonitrile solution of Cy5 (8) in a
ratio of 1:9 (v/v) (Table 1). When various concentrated PBS solutions
of Na ascorbate were added to an acetonitrile solution of 8, the flu-
orescence intensity did not change much. Therefore, the ratio of the
addition of a PBS solution of Na ascorbate to an acetonitrile solu-
tion of hybrid compounds was decided to be a 1:9 (v/v), when the
hybrid compound was reduced. The ESR and fluorescence spectra
of the synthesized bis-5-(TEMN-3-yl-carbonylamino)-Cy5 (12) and
-Cy3 (18) were measured before and after addition of a PBS solution
of Na ascorbate (Tables 2 and 3). The relative fluorescent intensity
(RFI) of hybrid compound 12 was 0.156 in comparison with Cy5 (8,
1.00) and RFI after reduction by the addition of Na ascorbate was
0.150. The RFI of the hybrid compound comprised of Cy3 (18) was
0.430 in comparison with Cy3 (14, 1.00); RFI after reduction was
0.256. No recovery of the fluorescence intensity of these hybrid
compounds was observed. The RFIs of 5-acetamido-Cy5 (11) and
-Cy3 (17) in which an acetyl group was introduced in the place
of TEMN, were also 0.09 and 0.22, respectively. Therefore, it was
assumed that the low fluorescence intensity was not due to the
introduction of nitroxide radicals, but, rather, to the amido-bond.
The RFIs of bis-5-nitro-Cy5 (9) and -Cy3 (15) were 0.564 and 4.51,
Scheme 1. Synthesis of hybrid compounds (12, 13, 18) by condensation of 5-bis-amino-Cy5 (10) or -Cy3 (16) and 3-carboxy-TEMN (7). Reagents and conditions—(a) CH3I
(2.0 equiv.) in toluene at r.t. 58 h; (b) NaNO3 (1.1 equiv.) in conc. H2SO4 at 0 ◦C, 0.5 h; (c) for Cy5: malonaldehydedianilide hydrochloride (0.5 equiv.) in AcOH/Ac2O, reflux
2 h; for Cy3: triethyl orthoformate (1.0 equiv.) in AcOH/Ac2O, reflux 1 h; (d) 14% aq. KI, reflux 1 h; (e) SnCl2·2H2O (12 equiv.) in conc. HCl, reflux 1 h; (f) Ac2O at r.t. 1 h; (g)
3-carboxy-TEMN (2.5 equiv.)/DCC (2.8 equiv.)/DMAP (0.30 equiv.) in CH3CN, at 0 ◦C, r.t. 15 h; (h) 1. hydrazobenzene (9.5 equiv.) in CH2Cl2 at r.t. 3 h; 2. Ac2O in CH2Cl2 at r.t. 6.5
h.

S. Sato et al. / Spectrochimica Acta Part A 71 (2009) 2030–2039
2033
Fig. 6. Fluorescence intensity of Cy5 (8) in some solvents (c 0.2 ␮M, ꢀex = 630 nm).
Table 1
Influence for the fluorescence intensity of Cy5 (8 in acetonitrile) after addition
of various concentrated solution of Na ascorbate in PBS (0.1 M, pH 7.0) (acetoni-
trile:PBS = 9:1, v/v)
Fig. 7. Changes in fluorescence (ꢀex 649 nm) and ESR spectra at 20 min after three
concentrations of Na ascorbate in PBS (a: 0 mM, b: 1.0 mM, c: 30 mM) were added
to a 20-␮M solution of 21 in PBS (pH 7.0) (1:9, v/v).
Solvent
Concentration of
Fluorescence intensity
Na ascorbate (␮M)
(c = 0.2 ␮M)
is more electron attracting (Fig. 5). The synthesis used the fol-
lowing reaction sequence as shown in Scheme 2: (1) synthesis
of N-methyl 5-carboxy-indolenium iodide; (2) synthesis of bis-5-
carboxy-Cy5 and -Cy3; and (3) introduction of 3-amino-PROXYL (6)
at the carboxyl group occupying the 5-position of the indolenine
moiety.
ESR and fluorescence intensities of the synthesized bis-5-
(PROXYL-3-yl-aminocarbonyl)-Cy5 (21) were measured before and
after the addition of 30 mM PBS solution of Na ascorbate (Fig. 7).
As expected, after addition of Na ascorbate, the RFI of 21 increased
to 1.80-fold with the decay in ESR intensity. The RFI (1.48) of dia-
magnetic hydroxylamine acetate of 21 (22) increased 2.22-fold in
comparison with that of paramagnetic 21 (0.668, Table 4). For Cy3,
after addition of Na ascorbate, the RFI of 24 increased 5.1-fold,
and the RFI of the diamagnetic hydroxylamine acetate (25) was
increased 6.54-fold compared with the hybrid compound com-
posed of Cy3 (24) (Table 4). Next, fluorescence intensity for the
hybrid compound 21 composed of Cy5, which is more unstable
CH3CN
–
0
8.70
7.60
CH3CN:Na ascorbate in PBS = 9:1
100
500
2,500
12,500
62,500
7.50
7.55
7.45
7.35
7.25
respectively, and neither bis-5-amino-Cy5 (10) nor -Cy3 (16) exhib-
ited any fluorescence. These results suggest that the fluorescence
intensities of Cy5 and Cy3 substituted with an electron-donating
group at the 5-position decrease significantly, while substitution
with an electron-withdrawing group results in a small decrease in
nitro-Cy5 and a large increase in nitro-Cy3.
The third strategy examined was introduction of the nitrox-
ide radical at the 5-position of both indolenines via substitution
of a carbonylamino group with an aminocarbonyl group, which
Table 2
Absorption and fluorescence spectra of Cy5 (8, 9, 10, 11), Cy5-nitroxide radicals hybrid compounds (12), and 12 + Na ascorbate
Compound
Absorption (in CH3CN)
Fluorescence (c 0.2 ␮M in CH3CN)
ꢀmax (nm)
log ε
ꢀex (nm)
ꢀem (nm)
RFI
8
9
10
11
637
661
694
666
669
–
5.33
5.32
5.15
5.23
5.34
–
634
654
–
662
665
668
650
670
–
682
682
680
1.00
0.564
–
0.094
0.156
0.150
12
12 + Na ascorbate
Table 3
Absorption and fluorescence spectra of Cy3 (14, 15, 16, 17), Cy3-nitroxide radicals hybrid compounds (18), and 18 + Na ascorbate
Compound
Absorption (in CH3CN)
Fluorescence (c 0.2 ␮M in CH3CN)
ꢀmax (nm)
log ε
ꢀex (nm)
ꢀem (nm)
RFI
14
15
16
17
542
565
596
573
575
–
5.10
5.21
4.91
4.90
5.08
–
540
562
–
572
572
572
556
577
–
596
596
598
1.00
4.51
–
0.22
0.43
0.26
18
18 + Na ascorbate

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S. Sato et al. / Spectrochimica Acta Part A 71 (2009) 2030–2039
Scheme 2. Synthesis of hybrid compounds (21, 22, 24, 25) by condensation of 5-bis-carboxy-Cy5 (19) or -Cy3 (23) and 3-amino-PROXYL (6). Reagent and conditions—(a)
1. 16% aq. NaNO2 in HCl at 0 ◦C, 0.5 h; 2. SnCl2·2H2O (3.0 equiv.) at 0 ◦C, 0.5 h; (b) 3-methyl-2-butanone/conc. H2SO4 in CH3CN, reflux 7 h; (c) CH3I (2.0 equiv.) in CHCl3,
reflux 7.5 h; (d) for Cy5: malonaldehydedianilide hydrochloride (0.5 equiv.) in AcOH/Ac2O, reflux 2 h, or 1,1,3,3-tetraethoxypropane (0.55 equiv.) in pyridine, reflux 10.5 h; for
Cy3: triethyl orthoformate (1.0 equiv.) in AcOH/Ac2O, reflux 1 h; (e) aniline (6.5 equiv.)/DCC (3.0 equiv.)/DMAP (0.6 equiv.) in CH3CN, at 0 ◦C–r.t., 19.5 h; (f) 1. hydrazobenzene
(10 equiv.) in CH2Cl2 at r.t. 1.5 h; 2. Ac2O in CH2Cl2, at r.t. 11 h.
Table 4
than one composed of Cy3 was measured under various pH con-
Relative fluorescence intensity of hybrid compounds (21, 24) and their hydroxy-
lamine acetates (22, 25)
ditions. Hybrid compound 21 was stable between pH 1 and 6, and
fluorescence intensity decreased gradually above pH 6, as shown
in Fig. 8. When a 15 mM PBS solution of Na ascorbate was added
(1:1, v/v) to a 20 ␮M PBS solution of 21, the ESR intensity decreased
immediately and the reaction was complete within 15 min (Fig. 9).
Compound Absorption (in CH3CN)
Fluorescence (c 0.2 ␮M in CH3CN)
ꢀex (nm) ꢀem (nm) RFI
ꢀmax (nm) log ε
8
19
20
21
22
24
25
637
649
653
650
650
555
556
5.33
5.31
5.06
5.36
5.37
5.05
5.23
634
650
652
649
647
555
552
650
658
664
661
661
568
568
1.00
0.084
0.80
0.67
1.48
1.00
6.54
The fluorescence intensity of 21 increased in parallel to the decay in
ESR (Fig. 10). However, ESR signals were not perfectly removed, that
may be due to the reoxidation of the corresponding hydroxylamine
by the resulting ascorbate radical [4]. When various concentrations
of the Na ascorbate in PBS were added to a 20 ␮M solution of 21 in
PBS, the ESR intensity of 21 decayed in proportion to the Na ascor-
bate concentration (Fig. 11), while the fluorescence intensity of 21
increased in parallel (Fig. 12).
Fig. 9. Time-course of the decay in ESR intensity after addition of 15-mM PBS solu-
tion of Na ascorbate to 20-␮M PBS solutions of 3-amino-PROXYL (6, ꢀ) and 21 (ꢁ)
(1:1, v/v), respectively.
Fig. 8. Effect of pH on the fluorescence intensity of 8 (1.0 ␮M, upper panel) and 21
(1.0 ␮M, lower panel).

S. Sato et al. / Spectrochimica Acta Part A 71 (2009) 2030–2039
2035
hybrids (24) fluoresced at 568 nm. Thus, these hybrid probes can be
used in the quantitative measurement of reducing species such as
Fe2+, ascorbic acid, and hydroxyl radicals. In addition, these hybrid
compounds can be used to visualize reducing species or radicals
in vivo. Similar to the phenomenon observed in hybrid compounds
composed of condensed-benzene ring fluorophores (naphthalene
and pyrene) and nitroxide radicals, fluorescence intensity of the
hybrid compounds composed of the conjugated polymethine fluo-
rophores and nitroxide radicals synthesized in this study increased
in parallel to the decrease in radicals. Maximum enhancement
of the fluorescence intensity was a 6.54-fold of the diamagnetic
hydroxylamine acetate (25).
4. Experimental
Fig. 10. Time-course of the increase in fluorescence intensity (0.2 ␮M, ꢀex 620 nm)
after addition of a 15-mM PBS solution of Na ascorbate to a 20-mM PBS solution of
21 (1:1, v/v).
4.1. Apparatus
Fluorescence spectra and relative fluorescence intensity were
measured using a Hitachi 650-10M fluorescence spectrophotome-
ter. Both the excitation and emission wavelength band passes were
both set at 2 nm and range was 1 and all concentrations of the
measured samples were 0.2 ␮M. Absorption spectra were obtained
using a Hitachi U-2010 UV-VIS spectrometer. Electron spin reso-
nance spectra were obtained on a JEOL JES-FR30 ESR spectrometer.
Samples were drawn into quartz capillaries, the bottoms of the cap-
illaries were sealed 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, 9.2 GHz; mod-
ulation amplitude, 1.25 G. All pH measurements were performed
using a Horiba M-13 PH meter.
Fig. 11. ESR intensity at 20 min after the addition of various concentrations of a PBS
solution of Na ascorbate to a 20-␮M PBS solution of 21 (1:1, v/v).
4.2. Synthesis
The solvents used in this study were purified by distilla-
tion. Reactions were monitored using TLC on 0.25-mm Silica Gel
F254 plates (E. Merck), and UV light and a 7% ethanolic solution
of phosphomolybdic acid with heat was used as the coloration
agents. Flash column chromatography was performed on silica
gel (230–400 mesh, Fuji-Silysia Co. Ltd., BW-300) to separate and
purify reaction products. 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 spectrom-
eter using KBr disks. NMR spectra were recorded on a Varian Inova
500 spectrometer using Me4Si as the internal standard. NMR spec-
tra of the hybrid compounds were measured as the corresponding
hydroxylamine induced by the reduction of nitroxyl radicals in the
presence of excess of hydrazobenzene. Mass spectral data were
obtained by fast-atom bombardment (FAB) using 3-nitrobenzyl
alcohol (NBA) as the matrix on a JEOL JMS-AX505HA instrument.
Fig. 12. Fluorescence intensity (0.2 ␮M, ꢀex 620 nm) at 20 min after the addition of
various concentrations of a PBS solution of Na ascorbate to a 20-␮M PBS solution of
21 (1:1, v/v).
4.2.1. Synthesis of nitroxide radicals
3. Conclusion
The four nitroxide radicals used in the present study (Fig. 3)
were synthesized by conventional methods [5], as shown in
Schemes 3 and 4.
Hybrid probes composed of polymethine-cyanine dyes and
nitroxide radicals were synthesized and the relationships between
their fluorescence and ESR intensities for the hybrid probes were
evaluated. Among the hybrid compounds synthesized in the
present study, polymethine-cyanine dye–nitroxide radical hybrid
probes synthesized by the introduction of a nitroxide radical at
the 5-position of the indolenine skeletons of Cy5 and Cy3 via an
aminocarbonyl bond (21 and 24) showed that fluorescence inten-
sity increased in parallel to a decay in ESR intensity. After reduction
of radicals by addition of Na ascorbate, hybrid probes composed
of Cy5 and PROXYL radicals (21) fluoresced at 661 nm, while Cy3
Scheme 3. Synthesis of 4-hydroxy-TEMPO (6). Reagents and conditions: (a) 30%
H2O2, EDTA, Na2WO4, in H2O, r.t. 1 day.

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S. Sato et al. / Spectrochimica Acta Part A 71 (2009) 2030–2039
Scheme 4. Synthesis of 3-amino-PROXYL (4) and 3-carboxy-TEMN (5). Reagents and conditions—(a) Br2 in AcOH, at r.t. 1 day; (b) 25% aq. NH3, KOH, at 0 ◦C; (c) 30% H2O2,
EDTA, NaWO4 in MeOH–H2O, at r.t., 21 h; (d) 10% aq. NaOH, reflux 1 day; (e) H2/10% Pd-C, in MeOH, at r.t., 17 h; (f) NaOH, Br2, H2O, KOH, at 0–70 ◦C, 3 h; (g) LiAlH4 (1.15 equiv.)
in THF, at 0 ◦C–r.t., for 24 h.
4.2.2. Synthesis of hybrid compounds (8–25)
(d, 2H, J 9.0 Hz, ArH × 2), 8.35 (dd, 2H, J 2.2, 8.8 Hz, ArH × 2), 8.50 (t,
2H, J 12.7, 13.4 Hz, –CH × 2), 8.62 (d, 2H, J 2.2 Hz, ArH × 2). 13C NMR
(DMSO-d6) ı 26.8 (CH3 × 2), 32.0 (N-CH3 × 2), 49.1 (C3 × 2), 106.2,
128.2, 156.3 (each polymethine-C × 2, ×1, ×2), 175.1 (C2 × 2), 111.7,
118.6, 125.6, 142.4, 144.3, 148.3 (indolenylbenzene × 2). FAB-MS
(m/z) 473 (M)+.
The synthesis of Cy5 and Cy3 (8, 14) and their derivatives (9–13
and 15–25) were carried out as shown in Schemes 1 and 2 [2-g,h].
To a solution of p-aminobenzoic acid in H2O and concentrated
HCl, which was cooled in an ice-bath, a 16% aqueous NaNO2 solu-
tion was added and resulting mixture was stirred for 0.5 h. A
mixture of SnCl2·2H2O (34.0 g, 0.151 mmol) and concentrated HCl
(35 mL) was then added, followed by stirring in the ice-bath for
0.5 h. After work-up, the resulting crude white powder, hydra-
dine hydrochloride (11.6 g), was used in the subsequent reaction
without any purification. To a solution of HCl salt of hydradine
and 3-methyl-2-butanone in CH3CN concentrated H2SO4 (2.5 mL)
was added and refluxed for 7 h. After work-up and purification by
silica-gel column chromatography (n-hexane–AcOEt), 5-carboxy-
indolenine was afforded as orange crystals in 31% yield from
p-aminobenzoic acid. 5-Carboxyindolenine and methyl iodide were
refluxed in CHCl3 for 7.5 h to give ammonium iodide as a white pow-
der in 32% yield. Methyl 5-carboxyindolenium iodide was refluxed
with malonaldehydedianilide hydrochloride (0.5 equiv.) in AcOH
and Ac2O for 2 h or with tetraethoxypropane (0.55 equiv.) in pyri-
dine for 10.5 h, followed by successive purification using silica-gel
column chromatography (CHCl3–MeOH–AcOH) to give the cor-
responding Cy5 as dark-green crystals in the yields of 74% and
66%, respectively. Methyl 5-carboxyindolenium iodide was refluxed
with triethyl orthoformate (1.5 equiv.) in pyridine for 2 h, followed
by successive purification using silica-gel column chromatography
(CHCl3–MeOH–AcOH) to give the corresponding Cy3 as a dark-blue
powder in 82% yield.
4.2.2.3. 5-Amino-2-[5-(5-amino-1,3,3-trimethyl-2,3-dihydro
-1H-indol-2-ylidene)pent-1,3-dienyl]-1,3,3-trimethyl-3H-indolium
iodide (10). Mp = >300 ◦C. IR ꢁ 3311, 3216, 1622, 1466, 1334, 1157,
1093 cm−1
.
1H NMR (DMSO-d6) ı1.60 (s, 12H, CH3 × 4), 3.49 (s, 6H,
CH3 × 2), 5.29 (br. s, 4H, NH2 × 2), 6.05 (d, 2H, J 14.2 Hz, –CH × 2),
6.35 (t, 1H, J 12.4 Hz, –CH ), 6.57 (dd, 2H, J 2.2, 8.5 Hz, ArH × 2), 6.73
(d, 2H, J 2.2 Hz, ArH × 2), 7.05 (d, 2H, J 8.5 Hz, ArH × 2), 8.06 (t, 2H, J
12.9, 13.2 Hz, –CH × 2). 13C NMR (DMSO-d6) ı 27.5 (CH3 × 4), 31.2
(N-CH3), 48.7 (C3 × 2), 101.9, 123.4, 150.5 (each polymethine-C × 2,
×1, ×2), 170.0 (C2 × 2), 108.2, 111.7, 113.0, 133.0, 142.6, 147.2 (each
2C, indolenylbenzene × 2). FAB-MS (m/z) 413 (M)+.
4.2.2.4. 5-Acetoamido-2-[5-(5-acetoamido-1,3,3-trimethyl-2,
3-dihydro-1H-indol-2-ylidene)pent-1,3-dienyl]-1,3,3,-trimethyl-
3H-indolium iodide (11). Mp = >300 ◦C. IR ꢁ 1675, 1621, 1508,
1473, 1370, 1161, 1099 cm−1 1H NMR (DMSO-d6) ı1.63 (s, 12H,
.
CH3 × 4), 2.04 (s, 6H, COCH3 × 2), 3.56 (s, 6H, CH3 × 2), 6.19 (d,
2H, J 13.9 Hz, –CH × 2), 6.47 (t, 1H, J 12.2, 12.4 Hz, –CH ), 7.30
(d, 2H, J 8.5 Hz, ArH × 2), 7.43 (dd, 2H, J 2.0, 8.5 Hz, ArH × 2), 7.86
(d, 2H, J 2.0 Hz, ArH × 2), 8.23 (t, 2H, J 12.9, 13.2 Hz, –CH × 2),
10.05 (s, 2H, NH × 2). 13C NMR (DMSO-d6) ı 24.2 (COCH3), 27.3
(CH3 × 4), 31.3 (N-CH3 × 2), 49.0 (C3 × 2), 103.2, 124.9, 153.0 (each
polymethine-C × 2, ×1, ×2), 168.4 (C O × 2), 172.3 (C2 × 2), 111.3,
113.6, 119.2, 136.8, 138.4, 141.5 (each 2C, indolenylbenzene × 2).
FAB-MS (m/z) 497 (M)+.
4.2.2.1. 2-[5-(1,3,3-Trimethyl-2,3-dihydro-1H-indol-2-
ylidene)pent-1,3-dienyl]-1,3,3-trimethyl-3H-indolium iodide (8).
Mp = 268–269 ◦C. IR ꢁ 1496, 1458, 1373, 1333, 1172, 1103 cm−1 1H
.
NMR (CDCl3) ı1.68 (s, 12H, CH3 × 4), 3.61 (s, 6H, CH3 × 2), 6.28
(d, 2H, J 13.9 Hz, –CH × 2), 6.55 (t, 1H, J 12.2, 12.4 Hz, –CH ),
7.25 (m, 2H, ArH × 2), 7.40 (m, 4H, ArH × 4), 7.62 (d, 2H, J 7.6 Hz,
ArH × 2), 8.33 (t, 2H, J 11.7, 14.6 Hz, –CH × 2). 13C NMR (CDCl3) ı
27.9 (CH3 × 4), 32.3 (N-CH3 × 2), 49.3 (C3 × 2), 103.8, 126.3, 153.7
(each polymethine-C × 2, ×1, ×2), 173.4 (C2 × 2), 110.3, 122.1, 125.1,
128.5, 140.9, 142.6 (each 2C, indolenylbenzene × 2). FAB-MS (m/z)
383 (M)+.
4.2.2.5. 5-(2,2,5,5-Tetramethyl-pyrroline-1-oxyl
-3-yl)carbonylamino-2-{5-[5-(2,2,5,5-tetramethyl-pyrroline-1-
oxyl-3-yl)carbonylamino-1,3,3-trimethyl-2,3-dihydro-1H-indol-2-
ylidene]pent-1,3-dienyl}-1,3,3,-trimethyl-3H-indolium iodide (12).
Mp = >300 ◦C. IR ꢁ 1662, 1618, 1508, 1466, 1331, 1159 cm−1 1H NMR
.
(DMSO-d6) ı(PROXYL moiety): 1.20, 1.29 (each s, 12H, CH3 × 8),
1.64 (s, 12H, CH3 × 4), 6.54 (s, 2H, –CH × 2). (Cy5 moiety): 3.57
(s, 6H, CH3 × 2), 6.19 (d, 2H, J 13.9 Hz, –CH × 2), 6.47 (t, 1H, J
12.2, 12.4 Hz, –CH ), 7.32 (d, 2H, J 8.5 Hz, ArH × 2), 7.97 (s, 2H,
ArH × 2), 8.23 (t, 2H, J 13.2 Hz, –CH × 2), 9.88 (s, 2H, NH × 2).
13C NMR (DMSO-d6) ı(PROXYL moiety): 25.1, 25.3 (each CH3 × 2),
66.9, 69.3 (C2, 5), 139.9 (C4), 140.0 (C3). (Indolenine moiety): 27.3
(CH3 × 4), 31.3 (N-CH3 × 2), 49.0 (C3 × 2), 103.2, 125.0, 153.1 (each
4.2.2.2. 5-Nitro-2-[5-(5-nitro-1,3,3-trimethyl-2,3-dihydro-1H-indol-
2-ylidene)pent-1,3-dienyl]-1,3,3-trimethyl-3H-indolium iodide (9).
Mp = >300 ◦C. IR ꢁ 1498, 1457, 1373, 1321, 1300, 1163, 1079 cm−1 1H
.
NMR (DMSO-d6) ı 1.77 (s, 12H, CH3 × 4), 3.68 (s, 6H, CH3 × 2), 6.49
(d, 2H, J 13.9 Hz, –CH × 2), 6.73 (t, 1H, J 12.2, 12.4 Hz, –CH ), 7.60

S. Sato et al. / Spectrochimica Acta Part A 71 (2009) 2030–2039
2037
polymethine-C × 2, ×1, ×2), 163.0 (C O × 2), 172.3 (C2 × 2), 111.1,
114.4, 120.0, 136.5, 138.6, 141.4 (each 2C, indolenylbenzene × 2).
FAB-MS (m/z) 745 (M)+.
moiety): 21.0, 26.0, 26.6, 27.5 (each 2C, CH3 × 8), 40.6 (C4,4ꢀ), 53.5
(C3,3ꢀ), 60.4, 65.4 (each 2C, C2,2ꢀ,5,5ꢀ). (Cy5 moiety): 27.2 (CH3 × 4),
31.5 (N-CH3 × 2), 48.9 (C3 × 2), 104.4 (C × 2, polymethine-C), 126.4
(C × 2, polymethine-C), 154.8 (polymethine-C), 165.8 (C O × 2),
174.0 (C2 × 2), 110.7, 121.7, 128.5, 130.7, 141.0, 145.2 (each 2C,
indolenylbenzene). FAB-MS (m/z) 749 (M)+.
4.2.2.6. 5-(1-Acetoxy-2,2,5,5-tetramethylpyrrolidine
-3-yl)carbonylamino-2-{5-[5-(1-acetoxy-2,2,5,
5-tetramethylpyrrolidine-3-yl)carbonylamino-1,3,3-trimethyl-2,
3-dihydro-1H-indol-2-ylidene]pent-1,3-dienyl}-1,3,3,-trimethyl-3H-
indolium iodide (13). Mp = 243–246 ◦C. IR ꢁ 2972, 2931, 1751, 1670,
4.2.2.10. 5-(1-Acetoxy-2,2,5,5-tetramethylpyrrolidine
-3-yl)aminocarbonyl-2-{5-[5-(1-acetoxy-2,2,5,
1618, 1508, 1470, 1363, 1331 cm−1
.
1H NMR (DMSO-d6) ı (pyrroline
5-tetramethylpyrrolidine-3-yl)aminocarbonyl-1,3,3-trimethyl-2,
3-dihydro-1H-indol-2-ylidene]pent-1,3-dienyl}-1,3,3,-trimethyl-3H-
indolium iodide (22). Mp = 227–229 ◦C. IR ꢁ 3371, 2970, 2931, 1757,
moiety): 1.28 (s, 12H, CH3 × 4), 1.31, 1.42 (each s, 6H, CH3 × 4),
6.58 (s, 2H, –CH × 2). (Cy5 moiety): 1.66 (s, 12H, CH3 × 4), 2.13
(s, 6H, COCH3 × 2), 3.60 (s, 6H, CH3 × 2), 6.22 (d, 2H, J 13.9 Hz,
–CH × 2), 6.49 (t, 1H, J 12.0, 12.4 Hz, –CH ), 7.36 (d, 2H, J 8.5 Hz,
ArH × 2), 7.60 (dd, 2H, J 2.0, 8.7 Hz, ArH × 2), 7.96 (d, 2H, J 2.0 Hz,
ArH × 2), 8.25 (t, 2H, J 12.9, 13.2 Hz, –CH × 2), 10.03 (s, 2H,
NH × 2). 13C NMR (DMSO-d6) ı (pyrroline moiety): 19.1 (COCH3),
22.6, 23.0, 27.6, 28.4 (CH3 × 8), 68.5, 71.0 (C2,2ꢀ,5,5ꢀ), 138.9 (C3,3ꢀ),
170.9 (COCH3). (Cy5 moiety): 27.6 (CH3 × 4), 31.3 (N-CH3), 49.0
(C3), 103.3 (polymethine-C × 2), 125.0 (polymethine-C), 153.1
(polymethine-C × 2), 162.4 (C O × 2), 172.4 (C2 × 2), 111.2, 114.6,
120.2, 136.3, 138.7, 141.4 (each 2C, indolenylbenzene × 2). FAB-MS
(m/z) 831 (M)+.
1647, 1506, 1462, 1356, 1333, 1165 cm−1 1H NMR (DMSO-d6)
.
ı(PROXYL moiety): 1.00, 1.16, 1.18, 1.23 (s, each 6H, CH3 × 8), 1.99
(m, 4H, CH2 × 2), 4.49 (br. s, 2H, CH– × 2). (Cy5 moiety): 1.72
(s, 12H, CH3 × 4), 2.08 (s, 6H, COCH3 × 2), 3.64 (s, 6H, CH3 × 2),
6.35 (d, 2H, J 13.4 Hz, –CH × 2), 6.61 (t, 1H, J 12.0 Hz, –CH ),
7.47 (d, 2H, J 8.5 Hz, ArH × 2), 7.84 (dd, 2H, J 1.5, 8.4 Hz, ArH × 2),
8.13 (s, 2H, ArH × 2), 8.24 (br. s, 2H, NH × 2), 8.40 (t, 2H, J 12.7,
13.4 Hz, –CH × 2). 13C NMR (DMSO-d6) ı(PROXYL moiety): 19.1
(COCH3), 24.9, 25.0, 26.1 (CH3 × 8), 40.3 (C4,4ꢀ), 54.1 (C3,3ꢀ), 62.0,
66.2 (C2,2ꢀ,5,5ꢀ), 170.8 (COCH3 × 2). (Cy5 moiety): 27.2 (CH3 × 4),
31.5 (N-CH3 × 2), 48.9 (C3 × 2), 104.4 (polymethine-C × 2), 126.4
(polymethine-C), 154.9 (polymethine-C × 2), 166.0 (C O × 2),
174.0 (C2 × 2), 110.7, 121.7, 128.5, 130.5, 141.1, 145.3 (each 2C,
indolenylbenzene). FAB-MS (m/z) 835 (M)+.
4.2.2.7. 5-Carboxy-2-[5-(5-carboxy-1,3,3-trimethyl-2,
3-dihydro-1H-indol-2-ylidene)pent-1,3-dienyl]-1,3,3,-trimethyl
-3H-indolium iodide (19). Mp = >300 ◦C. IR ꢁ 3409, 2978, 1676,
1508, 1473, 1362, 1333, 1163 cm−1
.
1H NMR (DMSO-d6) ı 1.68 (s,
4.2.2.11. 2-[3-(1,3,3-Trimethyl-2,3-dihydro-1H-indol-2-
12H, CH3 × 4), 3.62 (s, 6H, CH3 × 2), 6.34 (d, 2H, J 13.9 Hz, –CH × 2),
6.60 (t, 1H, J 12.2, 12.4 Hz, –CH ), 7.45 (d, 2H, J 8.5 Hz, ArH × 2), 7.97
(dd, 2H, J 1.5, 8.4 Hz, ArH × 2), 8.13 (d, 2H, J 1.5 Hz, ArH × 2), 8.38 (t,
2H, J 12.9, 13.2 Hz, –CH × 2). 13C NMR (DMSO-d6) ı 27.1 (CH3 × 4),
31.6 (N-CH3 × 2), 48.9 (C3 × 2), 104.8 (polymethine-C × 2), 126.9
(polymethine-C × 2), 155.4 (polymethine-C), 167.3 (C O × 2),
174.4 (C2 × 2), 111.1, 123.6, 127.2, 130.7, 141.4, 146.6 (each 2C,
indolenylbenzene × 2). FAB-MS (m/z) 471 (M)+.
ylidene)propenyl]-1,3,3-trimethyl-3H-indolium
iodide
(14).
Mp = 241–243 ◦C. IR ꢁ 1556, 1496, 1452, 1396, 1207, 1105 cm−1
.
1H NMR (DMSO-d6) ı1.69 (s, 12H, CH3 × 4), 3.65 (s, 6H, CH3 × 2),
6.47 (d, 2H, J 13.7 Hz, –CH × 2), 7.30 (m, 2H, ArH × 2), 7.45 (m, 4H,
ArH × 4), 7.64 (d, 2H, J 7.57 Hz, ArH × 2), 8.34 (t, 1H, J 13.4, 13.7 Hz,
–CH ). 13C NMR (CDCl3) ı 28.0 (CH3 × 4), 32.6 (N-CH3 × 2), 48.8
(C3 × 2), 104.8, 150.6 (each polymethine-C × 1, ×2), 174.2 (C2 × 2),
110.7, 122.0, 125.3, 128.8, 140.3, 142.5 (each 2C, indolenylbenzene).
FAB-MS (m/z) 357 (M)+.
4.2.2.8. 5-Anilidyl-2-[5-(5-anilidyl-1,3,3-trimethyl-2,3-dihydro-
1H-indol-2-ylidene)pent-1,3-dienyl]-1,3,3,-trimethyl-3H-indol
ium iodide (20). Mp = 154–158 ◦C. IR ꢁ 3047, 2929, 1728, 1647,
4.2.2.12. 5-Nitro-2-[3-(5-nitro-1,3,3-trimethyl-2,3-dihydro
-1H-indol-2-ylidene)propenyl]-1,3,3-trimethyl-3H-indolium iodide
1564, 1468, 1356, 1211, 1163 cm−1
.
1H NMR (DMSO-d6) ı 1.75 (s,
(15). Mp = >300 ◦C. IR ꢁ 1556, 1448, 1388, 1329, 1200, 1097 cm−1
.
12H, CH3 × 4), 3.65 (s, 6H, CH3 × 2), 6.45 (d, 2H, J 13.9 Hz, –CH × 2),
6.63 (t, 1H, J 12.4, 12.7 Hz, –CH ), 7.11 (m, 2H, aniline–ArH × 2), 7.36
(m, 4H, aniline–ArH × 4), 7.51 (d, 2H, J 8.3 Hz, indolenine–ArH × 2),
7.77 (d, 4H, J 7.6 Hz, aniline–ArH × 4), 8.06 (dd, 2H, J 1.5, 8.4 Hz,
indolenine–ArH × 2), 8.20 (d, 2H, J 1.5 Hz, indolenine–ArH × 2),
8.38 (t, 2H, J 12.9 Hz, –CH– × 2), 10.19 (s, 2H, NH × 2). 13C NMR
1H NMR (DMSO-d6) ı 1.77 (s, 12H, CH3 × 4), 3.74 (s, 6H, CH3 × 2),
6.73 (d, 2H, J 13.4 Hz, –CH × 2), 7.71 (d, 2H, J 8.8 Hz, ArH × 2), 8.40
(dd, 2H, J 2.2, 8.8 Hz, ArH × 2), 8.44 (t, 1H, J 13.4 Hz, –CH ), 8.62 (d,
2H, J 2.2 Hz, ArH × 2). 13C NMR (DMSO-d6) ı 27.2 (CH3 × 4), 32.4
(N-CH3 × 2), 49.3 (C3 × 2), 106.0, 151.9 (each polymethine-C × 2,
×1), 176.8 (C2 × 2), 112.4, 118.6, 125.7, 142.0, 144.9, 148.1 (each 2C,
indolenylbenzene). FAB-MS (m/z) 447 (M)+.
(DMSO-d6)
ı
27.2 (CH3 × 4), 31.6 (N-CH3 × 2), 48.9 (C3 × 2),
104.6 (polymethine-C × 2), 126.6 (polymethine-C × 1), 156.8
(polymethine-C × 2), 164.9 (C O × 2), 174.2 (C2 × 2), 110.8, 122.1,
123.9, 131.0, 141.2, 145.6 (each 2C, indolenylbenzene × 2), 107.1,
120.7, 128.8, 139.3 (aniline × 2). FAB-MS (m/z) 621 (M)+.
4.2.2.13. 5-Amino-2-[3-(5-amino-1,3,3-trimethyl-2,3-dihydro
-1H-indol-2-ylidene)propenyl]-1,3,3-trimethyl-3H-indolium iodide
(16). Mp = 243–246 ◦C. IR ꢁ 3321, 3226, 1616, 1560, 1471, 1390,
1356, 1211, 1167, 1107 cm−1 1H NMR (DMSO-d6) ı 1.60 (s, 12H,
.
4.2.2.9. 5-(2,2,5,5-Tetramethylpyrrolidine-1-oxyl
CH3 × 4), 3.51 (s, 6H, CH3 × 2), 5.32 (br. s, 4H, NH2 × 2), 6.16 (d,
2H, J 13.7 Hz, –CH × 2), 6.58 (dd, 2H, J 2.2, 8.5 Hz, ArH × 2), 6.73
(d, 2H, J 2.0 Hz, ArH × 2), 7.08 (d, 2H, J 8.3 Hz, ArH × 2), 8.09 (t,
1H, J 13.5 Hz, –CH ). 13C NMR (DMSO-d6) ı 27.8 (CH3 × 4), 31.2
(N-CH3 × 2), 48.7 (C3 × 2), 101.1, 145.6 (each polymethine-C × 2,
×1), 170.8 (C2 × 2), 108.1, 112.0, 113.1, 132.8, 142.1, 147.5 (each 2C,
indolenylbenzene × 2). FAB-MS (m/z) 387 (M)+.
-3-yl)aminocarbonyl-2-{5-[5-(2,2,5,5-tetramethylpyrrolidine
-1-oxyl-3-yl)aminocarbonyl-1,3,3-trimethyl-2,3-dihydro-1H-indol-
2-ylidene]pent-1,3-dienyl}-1,3,3,-trimethyl-3H-indolium iodide (21).
Mp = 273–276 ◦C. IR ꢁ 3400, 2972, 2931, 1647, 1604, 1506, 1462,
1356, 1333, 1165 cm−1 1H NMR (DMSO-d6) ı(PROXYL moiety):
.
0.93, 1.07 (each s, 6H, CH3 × 4), 1.13 (s, 12H, CH3 × 4), 1.80, 1.89
(each m, 2H, CH2 × 2), 4.34 (m, 2H, CH– × 2). (Cy5 moiety): 1.70 (s,
12H, CH3 × 4), 3.62 (s, 6H, CH3 × 2), 6.31 (d, 2H, J 13.7 Hz, –CH × 2),
6.57 (t, 1H, J 12.2 Hz, –CH ), 7.44 (d, 2H, J 8.3 Hz, ArH × 2), 7.96
(d, 2H, J 8.3 Hz, ArH × 2), 8.14 (s, 2H, ArH × 2), 8.18 (s, 2H, NH × 2),
8.37 (t, 2H, J 12.9 Hz, –CH × 2). 13C NMR (DMSO-d6) ı(PROXYL
4.2.2.14. 5-Acetoamino-2-[3-(5-acetoamino-1,3,3-trimethyl-2,
3-dihydro-1H-indol-2-ylidene)propenyl]-1,3,3,-trimethyl-3H-
indolium iodide (17). Mp = >300 ◦C. IR ꢁ (KBr) 3251, 3209, 3080,
1676, 1624, 1560, 1466, 1394, 1201, 1115 cm−1 1H NMR (DMSO-d6)
.

2038
S. Sato et al. / Spectrochimica Acta Part A 71 (2009) 2030–2039
ı 1.64 (s, 12H, CH3 × 4), 2.05 (s, 6H, COCH3 × 2), 3.60 (s, 6H,
CH3 × 2), 6.37 (d, 2H, J 13.7 Hz, –CH × 2), 7.37 (d, 2H, J 8.5 Hz,
ArH × 2), 7.54 (dd, 2H, J 1.7, 8.5 Hz, ArH × 2), 7.83 (d, 2H, J 2.0 Hz,
(d, 2H, J 13.4 Hz, –CH × 2), 7.54 (d, 2H, J 8.5 Hz, ArH × 2), 7.99
(dd, 2H, J 1.0, 8.3 Hz, ArH × 2), 8.11 (s, 2H, ArH × 2), 8.29 (br. s, 2H,
NH × 2), 8.36 (t, 1H, J 13.4, 13.7 Hz, –CH ). 13C NMR (DMSO-d6)
ı(PROXYL moiety): 19.1, 170.8 (OAc × 2), 26.1 (br, CH3 × 4), 40.7
(br, C4 × 2), 53.9 (br, C3 × 2), 61.9, 66.3 (C2, 5 × 2). (Cy3 moiety):
27.4 (CH3 × 4), 31.9 (N-CH3 × 2), 49.0 (C3 × 2), 104.1, 150.4 (each
polymethine-C × 2, ×1), 111.2, 121.9, 128.6, 131.2, 140.7, 145.2 (each
2C, indolenylbenzene × 2), 166.0 (C O × 2), 175.5 (C2 × 2). FAB-MS
(m/z) 809 (M)+.
ArH × 2), 8.24 (t, 1H, J 13.4, 13.7 Hz, –CH ), 10.16 (s, 2H, NH × 2). 13
C
NMR (DMSO-d6) ı 24.2 (COCH3), 27.6 (CH3 × 4), 31.5 (N-CH3 × 2),
49.0 (C3 × 2), 102.6 (polymethine-C × 2), 148.4 (polymethine-C),
168.5 (C O × 2), 173.4 (C2 × 2), 111.7, 113.6, 119.4, 137.2, 138.2,
141.1 (each 2C, indolenylbenzene × 2). FAB-MS (m/z) 471 (M)+.
4.2.2.15. 5-(2,2,5,5-Tetramethylpyrroline-3-yl)carbonylamino
-2-{3-[5-(2,2,5,5-tetramethylpyrroline-3-yl)carbonylamino-1,3,
3-trimethyl-2,3-dihydro-1H-indol-2-ylidene]propenyl}-1,3,3,
4.3. Preparation of stock solutions
-trimethyl-3H-indolium iodide (18). Mp = >300 ◦C. IR
ꢁ
3298,
Stock solutions (0.001 M) of Cy5 and Cy3 derivatives 8–25 were
prepared in 10 mL of CH3CN and were kept at 4 ◦C in a refrigerator.
Buffer solutions in the pH range of 5.6–8.0 were prepared using
0.1 M PBS, while buffer solutions with pH values ranging from 1.09
to 5.5werepreparedusing1 M AcONaand1 M HCl. Each pHsolution
was prepared to the concentration of 20 ␮M by each buffer solution.
2973, 1672, 1624, 1560, 1454, 1390, 1205, 1107 cm−1
.
1H NMR
(DMSO-d6) ı(PROXYL moiety): 1.20, 1.29 (each s, 12H, CH3 × 8),
6.61 (s, 2H, –CH × 2). (Cy3 moiety): 1.65 (s, 12H, CH3 × 4), 3.60 (s,
6H, CH3 × 2), 6.36 (d, 2H, J 13.7 Hz, –CH × 2), 7.38 (d, 2H, J 8.5 Hz,
ArH × 2), 7.68 (dd, 2H, J 1.7, 8.7 Hz, ArH × 2), 7.93 (d, 2H, J 1.7 Hz,
ArH × 2), 8.24 (t, 1H, J 13.2, 13.7 Hz, –CH ), 9.99 (s, 2H, NH × 2). 13
C
NMR (DMSO-d6) ı(PROXYL moiety): 25.2, 25.4 (each CH3 × 2), 67.0,
69.4 (C2, 5), 139.8 (C4), 140.3 (C3). (Cy3 moiety): 27.6 (CH3 × 4),
31.6 (N-CH3 × 2), 49.1 (C3 × 2), 102.8 (polymethine-C × 2), 148.4
(polymethine-C), 163.2 (C O), 173.5 (C2 × 2), 111.6, 114.6, 120.4,
137.0, 138.5, 141.0 (each 2C, indolenylbenzene × 2). FAB-MS (m/z)
719 (M)+.
4.4. Fluorescence measurement
The above stock solutions were diluted with acetonitrile to
0.2 ␮M and subjected to the measurement.
Fluorescence measurement of the hybrid compounds after
reduction with Na ascorbate was as follows. A 20-␮M PBS solution
of the hybrid compound and a 15-mM PBS solution of Na ascorbate
were mixed in a ratio of 1:1 (v/v). And after the recognition of the
reduction of the radicals by ESR measurement, this sample solution
was diluted with acetonitrile to 0.2 ␮M and then was subjected to
the measurement of the fluorescence intensity.
4.2.2.16. 5-Carboxy-2-[3-(5-carboxy-1,3,3-trimethyl-2,3-dihydro
-1H-indol
-2-ylidene)propenyl]-1,3,3,-trimethyl-3H-indolium
iodide
(23).
Mp = >300 ◦C. IR ꢁ (KBr) 3429, 2970, 2929, 1720, 1556, 1469, 1392,
1193, 1105 cm−1
.
1H NMR (DMSO-d6) ı 1.71 (s, 12H, CH3 × 4), 3.67
(s, 6H, CH3 × 2), 6.57 (d, 2H, J 13.4 Hz, –CH × 2), 7.55 (d, 2H, J
8.5 Hz, ArH × 2), 8.03 (dd, 2H, J 1.5, 8.3 Hz, ArH × 2), 8.15 (d, 2H, J
1.5 Hz, ArH × 2), 8.38 (t, 1H, J 13.4 Hz, –CH ). 13C NMR (DMSO-d6)
ı27.4 (CH3 × 4), 32.0 (N-CH3 × 2), 49.0 (C3 × 2), 104.5, 150.9 (each
polymethine-C × 2, ×1), 111.7, 123.5, 127.8, 130.8, 141.0, 146.4 (each
2C, indolenylbenzene × 2) FAB-MS (m/z) 445 (M)+.
4.5. ESR measurement
Sample solutions were prepared with 0.1 M PBS (pH 7.0) to
20 ␮M and were subjected to the measurement of the ESR. This 20-
␮M PBS sample solution and a 15-mM PBS of Na ascorbate were
mixed in a ratio of 1:1 (v/v) for reduction of the nitroxide radicals
and the resulting mixed solution was subjected to the measure-
ment of the ESR.
4.2.2.17. 5-(2,2,5,5-Tetramethylpyrrolidine-1-oxyl-3
-yl)aminocarbonyl-2-{3-[5-(2,2,5,5-tetramethylpyrrolidine-1
-oxyl-3-yl)aminocarbonyl-1,3,3-trimethyl-2,3-dihydro-1H-indol-
Acknowledgements
2-ylidene]propenyl}-1,3,3,-trimethyl-3H-indolium
Mp = >300 ◦C. IR ꢁ 3400, 2974, 2933, 1651, 1562, 1458, 1388, 1211,
1112 cm−1 1H NMR (DMSO-d6) ı(PROXYL moiety): 0.96, 1.08,
iodide
(24).
The authors wish to thank Professors Tateaki Ogata for the use
of a JEOL JES-FR30 ESR spectrometer, and Mr. Hiroya Masukawa and
Mr. Yuki Takaoka for their technical assistance.
.
1.14, 1.15 (each s, 6H, CH3 × 8), 1.83, 1.92 (each m, 2H, CH2 × 2),
4.37 (m, 2H, CH– × 2). (Cy3 moiety): 1.707, 1.710 (each s, 6H,
CH3 × 4), 3.67 (s, 6H, CH3 × 2), 6.52 (d, 2H, J 13.4 Hz, –CH × 2),
7.25 (br. s, 2H, OH × 2), 7.53 (d, 2H, J 8.3 Hz, ArH × 2), 8.00 (dd,
2H, J 1.5, 8.3 Hz, ArH × 2), 8.16 (d, 2H, J 1.5 Hz, ArH × 2), 8.33 (s,
2H, NH × 2), 8.35 (t, 1H, J 13.4 Hz, –CH ). 13C NMR (DMSO-d6)
ı(PROXYL moiety): 20.7, 25.7, 26.3, 27.1 (CH3 × 4), 40.3 (C4), 53.2
(C3), 60.0 and 65.0 (C2, 5). (Cy3 moiety): 27.3 (CH3 × 4), 31.6
(N-CH3 × 2), 48.7 (C3 × 2), 103.7, 155.8 (each polymethine-C × 2,
×1), 165.6 (C O × 2), 175.1 (C2 × 2), 110.9, 121.6, 128.3, 131.1, 140.3,
144.7 (each 2C, indolenylbenzene × 2). FAB-MS (m/z) 723 (M)+.
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