Novel membrane-localizing TEMPO derivatives for measurement of cellular oxidative stress at the cell membrane

Article abstract of DOI:10.1016/j.bmcl.2006.11.040

Oxidative stress affecting lipid membranes is considered to be closely related to cardiovascular disease and brain ischemia. In this study, we designed and synthesized membrane-localizing TEMPO derivatives and demonstrated that one of these synthesized probes, compound 1, localized and detected oxidative stress in the cell membrane in an endotoxic model of a mouse macrophage-like cell line. Compound 1 is therefore a potentially useful probe for evaluating oxidative stress at the cell membrane.

Full text of DOI:10.1016/j.bmcl.2006.11.040

Bioorganic & Medicinal Chemistry Letters 17 (2007) 1451–1454
Novel membrane-localizing TEMPO derivatives for measurement
of cellular oxidative stress at the cell membrane
Shizuka Ban, Hidehiko Nakagawa,* Takayoshi Suzuki and Naoki Miyata*
Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi 467-8603, Japan
Received 22 August 2006; revised 30 October 2006; accepted 13 November 2006
Available online 16 November 2006
Abstract—Oxidative stress affecting lipid membranes is considered to be closely related to cardiovascular disease and brain ischemia.
In this study, we designed and synthesized membrane-localizing TEMPO derivatives and demonstrated that one of these synthesized
probes, compound 1, localized and detected oxidative stress in the cell membrane in an endotoxic model of a mouse macrophage-like
cell line. Compound 1 is therefore a potentially useful probe for evaluating oxidative stress at the cell membrane.
ꢀ 2006 Elsevier Ltd. All rights reserved.
It is known that about 1% of the oxygen taken into our
bodies by breathing is metabolized to reactive oxygen
species (ROS). ROS are considered to play important
roles, not only in inflammation as protective factors,
but also in signal transduction.1 On the other hand, it
is also known that ROS react with lipids, proteins, sug-
ars, and DNA, causing oxidative stress when produced
in excess.2 These reactions are suggested to be a major
cause of a variety of diseases and oxidative stress caused
by their reaction with lipids is considered to be closely
related to cardiovascular disease3 and brain ischemia.4
It appears important to evaluate oxidative stress with
respect to lipids in order to understand the pathological
conditions underlying these kinds of diseases. However,
there have been only a few attempts to measure the oxi-
dative stress induced by ROS in specific cellular
regions.5 ROS can be measured indirectly by means of
their reaction with stable radical compounds in the cell,
through which the radical probes are readily reduced to
non-radical species.6
downregulated. Electron spin resonance (ESR) measure-
ment is a useful approach to detect radical species in
biological systems. Using TEMPOL (4-hydroxyl-
2,2,6,6-tetramethyl-piperidin-1-oxyl), a useful TEMPO
derivative, oxidative stress can be measured by ESR
spectrometry.
TEMPOL is easily introduced into cells, but due to its
amphiphilic nature can easily exit from cells as well.8
TEMPO derivatives which localize to a particular cellu-
lar region would be useful for measuring oxidative stress,
including stress on the cell membrane, and TEMPO
derivatives localizing to the lipid membrane would be
advantageous.
For this purpose, the TEMPO derivatives would require
a radical moiety for ESR detection, a fluorescent group
for identifying its cellular distribution, and a functional
group for localizing to the lipid membrane in a cell.
In this study, we designed and synthesized TEMPO
derivatives with an alkyl chain for localization to the cell
membrane (Fig. 1), and demonstrated that these radical
probes were able to detect oxidative stress in lipid bilay-
ers in an endotoxic model of a mouse macrophage-like
cell line.
Among these radical species, 2,2,6,6-tetramethylpiperi-
din-1-oxyl (TEMPO) can be reduced to 2,2,6,6-tetrame-
thylpiperidin-1-ol under physiological conditions, that
is, TEMPO converts to the non-radical form by reduc-
tion.7 When ROS are upregulated and cells are in a rel-
atively oxidative environment, cellular reduction will be
Mouse RAW264.7 cells were cultured in DMEM con-
taining penicillin and streptomycin, supplemented with
fetal bovine serum. For the experiments, the cells were
plated onto 10-cm culture dishes at 1.5 · 107 cells/dish
with 15 mL DMEM. The cells were incubated at 37 ꢁC
Keywords: Fluorescein; Redox; Electron spin resonance; Reactive ox-
ygen species; Superoxide; Inflammation.
*
Corresponding authors. Tel.:/fax: +81 52 836 3407; e-mail addresses:
deco@phar.nagoya-cu.ac.jp; miyata-n@phar.nagoya-cu.ac.jp
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.11.040

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S. Ban et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1451–1454
Figure 1. Structures of TEMPO derivatives designed to localize to cell membranes.
0.5 lg/mL) and IFN-c (human recombinant, 150 U/mL).
The treated cells were subsequently cultured for 5 h,
washed 2 times with Dulbecco’s PBS (D-PBS), and
then treated with 100 lM of 1 for 10 min in dark.
Following this, they were washed 3 times with D-PBS.
The cells were then scraped into 2 mL D-PBS, and
1 mL of the cell suspension was used for ESR
experiments.
Each suspension of treated cells was placed in a flat
quartz cuvette. ESR measurements were started 15 min
after the treatment with 1. The ESR signal was recorded
at 5-min intervals. The signal intensity (I) was calculated
from the 2nd integral of the signal trace and expressed as
a ratio (I/I0) by comparing it to the intensity of the stan-
dard Mn2+ signal (I0). The signal decay rate was calcu-
lated as the pseudo first order rate of the decrease in
the ratio (I/I0).
Figure 2. Distribution of 1 in RAW264.7 cells. The cells were stained
with 1 (green) and Hoechst 33342 (blue), and then observed by
confocal fluorescence microscopy.
For confocal microscopy, the cells were plated on a 3-cm
glass-bottomed culture dish at 1.5 · 105cells/dish with
1.5 mL DMEM, and incubated at 37 ꢁC in a humidified
5% (v/v) CO2 incubator for 2 days. The cells were treated
with 1 or 2 in the same manner as for the ESR experi-
in a humidified 5% (v/v) CO2 incubator for 2 days. Then,
the culture medium was replaced with 5 mL of serum-
free DMEM, and the cells were treated with LPS (E. coli,
Scheme 1. Synthesis of compound 1. Reagents and conditions: (a) n-decanonyl chloride, Et3N, CH2Cl2, 99%; (b) Pd–C, H2, MeOH, then 4 N HCl/
AcOEt, 88%; (c) FITC, Et3N, EtOH, 51%; (d) LiOH aq, THF, EtOH, 98%; (e) 4-amino-TEMPO, EDCI, HOBt, DMF, 55%.

S. Ban et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1451–1454
1453
ments. The cells were subsequently stained with Hoechst
33342 for 10 min and subjected to confocal fluorescence
microscopy.
etrate into the lipid bilayer for its adequate lipid solubil-
ity. Compounds 1 and 2 are assumed to be distributed at
the both leaflets of the cell membrane. Thus, the probe
can measure the oxidative stress at the cell membrane
(Scheme 1 and Scheme 2).
The confocal microscopic study of the RAW264.7 cells
treated with compound 1 indicated that 1 was localized
to the cell membrane as expected (Fig. 2), due to the
presence of its alkyl chain. The TEMPO moiety was as-
sumed to be close to the surface in the lipid bilayer be-
cause the signal shape was not broad, as compared
with the signal of TEMPOL in solution (Supporting
information). This probe is considered to be able to pen-
There are no differences in localization and sensitivity
between compounds 1 and 2 in the observation by con-
focal fluorescence microscopy. Compound 1 was used
for ESR analysis because it was more stable than com-
pound 2 in solution. The ESR signal of 1 was measured
in RAW264.7 cells to evaluate oxidative stress in the cell
Scheme 2. Synthesis of compound 2. Reagents and conditions: (a) n-nonylamine, THF, then NaBH(OAc)3, 84%; (b) FITC, THF, 41%.
Figure 3. The time course of the relative signal intensity measured at 5-min intervals, (a) control cells, (b) LPS/IFN-c-treated cells, and (c) LPS/IFN-
c-treated cells in the presence of SOD and catalase. I, compound 1 peak area; I0, Mn2+ external standard peak area. (d) Signal decay rate of 1 in
RAW264.7 cells. The signal decay rates of 1 with RAW264.7 cells were calculated from ESR signal intensities of 1 in RAW264.7 cells treated with
vehicle, or with LPS/IFN-c in the presence or absence of SOD/catalase. Values are presented as means SD of 3 or 4 experiments. ANOVA and
Bonferroni-type multiple t-test indicated significant differences between LPS/IFN-c and the control (**P < 0.01), and LPS/IFN-c + SOD/catalase
(*P < 0.05).

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S. Ban et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1451–1454
membrane. In the control cells, the signal intensity of 1
was gradually decreased at 0.0091 0.002 minÀ1 under
our conditions. The upregulation of oxidative stress
was evaluated after endotoxic stimulation. ESR spectra
of 1 were measured in RAW264.7 cells treated with
500 ng/mL LPS and 150 U/mL IFN-c for 5 h. The rate
of signal decay observed in cells treated with LPS/
IFN-c was decreased to 0.0049 0.0007 minÀ1. The de-
creased rate as a result of the LPS/IFN-c treatment was
restored to 0.0072 0.0004 minÀ1 in the presence of
100 U/mL SOD and 10 U/mL catalase during measure-
ment (Fig. 3d). In the absence of the RAW264.7 cells,
the signal failed to decay (data not shown).
and reduced in the cell membrane. The direct oxidation
of TEMPO itself by superoxide probably does not affect
the signal decay rate under the condition used for these
measurements.
In conclusion, although there remain some detailed
characteristics to be clarified, compound 1 was found
to be a useful probe for evaluating oxidative stress at
the cell membrane.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.
2006.11.040.
Since cells generally exist in a reductive environment,
compound 1 was found to be gradually reduced to the
non-radical species in the presence of the control cells.
Treatment with LPS/IFN-c activated the cells and in-
creased the production of reactive oxygen and nitrogen
species (ROS/RNS). LPS/IFN-c treatment also de-
creased the decay rate of nitroxyl radical. The decrease
in this rate was recovered in the presence of 100 U/mL
SOD and 10 U/mL catalase. Although ROS were still
upregulated by LPS/IFN-c treatment, they were consid-
ered to be at least partially scavenged by SOD and cat-
alase. The probe was reduced under any conditions in
our experiment, and the decay rate was considered to re-
flect the cellular local reducing ability for the probe,
which would correlate with the oxidative stress in the
cell membrane. Thus, the decay rate of 1 was not as-
sumed to reflect specific ROS production, but the local
oxidant upregulation, although the production of super-
oxide was upregulated under this experimental condi-
tion, and superoxide, hydrogen peroxide, and hydroxyl
radical were considered to play an important role in this
result. It was suggested that the cell membrane was ex-
posed to an oxidative environment due to the increase
in ROS by LPS/IFN-c-pretreatment. Although SOD
and catalase were thought not to be distributed inside
the cells, they were able to get close to the cell mem-
brane, where compound 1 was localized. These two en-
zymes may contribute to the scavenging of ROS around
the cell membrane, and this is consistent with the fact
that ESR signal decay rate was restored under these con-
ditions. The change in this rate was assumed to be due to
either a decrease in the cellular reductants by ROS
upregulation or an increase in the oxidation of hydrox-
ylamine, a reduced form of 1.9 The TEMPO moiety can
be oxidized to the ESR-silent oxonium cation form by
superoxide.10 However, this cation was found to be rap-
idly reduced back to TEMPO by the superoxide itself.10
Compound 1 was considered to be repeatedly oxidized
References and notes
1. (a) Irani, K.; Goldschmidt-Clermont, P. J. Biochem.
Pharmacol. 1998, 55, 1339; (b) Irani, K.; Xia, Y.; Zweier,
J. L.; Sollott, S. J.; Der, C. J.; Fearon, E. R.; Sundaresan,
M.; Finkel, T.; Goldschmidt-Clermont, P. J. Science 1997,
275, 1649.
2. Darley-Usmar, V.; Halliwell, B. Pharm. Res 1996, 13,
649.
3. (a) Ballinger, S. W. Free Radic. Biol. Med. 2005, 38, 1278;
(b) Molavi, B.; Mehta, J. L. Curr. Opin. Cardiol. 2004, 19,
488.
4. (a) Love, S. Brain Pathol. 1999, 9, 119; (b) Sweeney, M. I.;
Yager, J. Y.; Walz, W.; Juurlink, B. H. Can. J. Physiol.
Pharmacol. 1995, 75, 1525.
5. (a) Wipf, P.; Xiao, J.; Jiang, J.; Belikova, NA.; Tyurin, V.
A.; Fink, M. P.; Kagan, V. E. J. Am. Chem. Soc. 2005,
127, 12460; (b) Okamoto, A.; Inasaki, T.; Saito, I. Bioorg.
Med. Chem. Lett 2004, 14, 3415; (c) Freedman, J. E.;
Keaney, J. F., Jr. Methods Enzymol. 1999, 301, 61.
6. Nakagawa, H.; Moritake, T.; Tsuboi, K.; Ikota, N.;
Ozawa, T. FEBS Lett. 2000, 471, 187.
7. (a) May, J. M.; Qu, Z. C.; Juliao, S.; Cobb, C. E. Free
Radic. Res. 2005, 39, 195; (b) Samuni, A.; Goldstein, S.;
Russo, A.; Mitchell, J. B.; Krishna, M. C.; Neta, P. J. Am.
Chem. Soc. 2002, 124, 8719; (c) Iannone, A.; Bini, A.;
Swartz, HM.; Tomasi, A.; Vannini, V. Biochem. Pharma-
col. 1989, 38, 2581.
8. Cuzzocrea, S.; McDonald, M. C.; Mazzon, E.; Dugo, L.;
Lepore, V.; Fonti, M. T.; Ciccolo, A.; Terranova, M. L.;
Caputi, A. P.; Thiemerann, C. Eur. J. Pharmacol. 2000,
406, 127.
9. Zigler, J. S., Jr.; Qin, C.; Kamiya, T.; Krishna, MC.;
Cheng, Q.; Tumminia, S.; Russell, P. Free Radic. Biol.
Med. 2003, 35, 1194.
10. Krishna, MC.; Russo, A.; Mitchell, JB.; Goldstein, S.;
Dafni, H.; Samuni, A. J. Biol. Chem. 1996, 271, 26026.