A novel mitochondria-localizing nitrobenzene derivative as a donor for photo-uncaging of nitric oxide

Article abstract of DOI:10.1016/j.bmcl.2011.02.027

We report a novel green-fluorescent NO donor, NBDNO, bearing a 2,6-dimethylnitrobenzene moiety for photocontrollable NO release and a triphenylphosphonium moiety for targeting to mitochondria. Photorelease of NO from NBDNO was confirmed by means of ESR an

Full text of DOI:10.1016/j.bmcl.2011.02.027

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2000–2002
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A novel mitochondria-localizing nitrobenzene derivative as a donor
for photo-uncaging of nitric oxide
Taeko Horinouchi a, Hidehiko Nakagawa a,c, , Takayoshi Suzuki a, Kiyoshi Fukuhara b, Naoki Miyata a,b,
⇑
⇑
a Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi 467-8603, Japan
b Organic Chemistry National Institute of Health Sciences, Setagaya, Tokyo 158-8501, Japan
c PRESTO, Japan Science and Technology Agency (JST), 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We report a novel green-fluorescent NO donor, NBDNO, bearing a 2,6-dimethylnitrobenzene moiety
for photocontrollable NO release and a triphenylphosphonium moiety for targeting to mitochondria.
Photorelease of NO from NBDNO was confirmed by means of ESR analysis in aqueous solution. Intracel-
lular release of NO from NBDNO was confirmed by using DAR-4M AM, an NO-specific fluorescence probe.
NBDNO was colocalized with MitoRed, a mitochondrial stain, in HCT116 colon cancer cells. Our results
indicate that NBDNO is an effective NO donor for time-controlled, mitochondria-specific NO treatment.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 24 December 2010
Revised 4 February 2011
Accepted 5 February 2011
Available online 12 February 2011
Keywords:
Nitric oxide
Photoinduced release
Mitochondria
Electron spin trapping
Nitric oxide (NO), although it exists as an unstable free radical
under ambient conditions, is involved in the maintenance and reg-
ulation of many vital functions.1 For example, it serves as a pleiotro-
pic bioregulator of blood pressure, a neuromodulator, and a
biodefence agent. Consequently, many NO donors have been devel-
oped and employed for biological studies.2 Most NO donors cur-
rently used, such as NOCs3 and NORs,4 release NO by spontaneous
autolysis. NO donors that permit spatially and temporally control-
lable NO release are expected to be useful for a variety of biological
studies.
Photoirradiation on/off systems for temporal control of NO
release are easy to use and serve to minimize the effect of irradia-
tion on living cells.5 Although sodium nitroprusside (SNP) is avail-
able as a photoinducible NO donor, it has the drawback of potential
toxicity due to the cyanide ligands.6 We have previously reported
4-substituted 2,6-dimethylnitrobenzenes as a new type of NO do-
nor.7 Ultraviolet A (UV-A) irradiation of 2,6-dimethylnitrobenzenes
and NO induces cellular apoptosis via mitochondria-dependent
pathways, mitochondria are a potential target for NO donors to ini-
tiate apoptotic cancer cell death.9 Furthermore, it was recently re-
ported that mitochondrial fission is induced by S-nitrosylated Drp1
protein,10 and this mechanism may be involved in Altzheimer dis-
ease.11 Mitochondria are also known to have nitric oxide synthase
activity.12 Therefore, mitochondria-specific NO donors would be
useful to elucidate NO functions in mitochondria.
With the aim of developing functional photoactivatable NO do-
nors, we designed and synthesized NBDNO (Fig. 1), bearing a 2,6-
dimethylnitrobenzene moiety for photocontrollable NO release, a
triphenylphosphonium13 moiety for targeting to mitochondria
and a fluorescent 7-nitrobenz-2-oxa-1,3-diazole14 moiety for visu-
having an extended p-electron system at the 4-position results in
efficient NO release as a result of rearrangement to arylnitrite.
We have also demonstrated that our 2,6-dimethylnitrobenzene
derivatives work as photocontrollable NO donors in HCT116 hu-
man colon cancer cells. Because the concentration and biological
influence of NO differ among organelles, organelle-specific photo-
controllable NO donors would be extremely useful.8 Since the
mitochondrion is a key organelle for apoptotic signal transduction,
⇑
Corresponding authors. Tel./fax: +81 52 836 3407 (H.N.).
E-mail address: deco@phar.nagoya-cu.ac.jp (H. Nakagawa).
Figure 1. Chemical structure of NBDNO (1)
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.02.027

T. Horinouchi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2000–2002
2001
alizing the compound by means of microscopy. The triphenylphos-
phonium moiety is expected to target mitochondria effectively due
to its hydrophobic cationic character. Furthermore, an NO donor
bearing a NBD green fluorescent moiety is expected to be useful
in multi-color labeling experiments, because many mitochondria-
localizing compounds have red fluorescence. Here, we show that
our novel NO donor, NBDNO, is distributed exclusively to mito-
chondria, where it releases NO in response to UV-A irradiation.
Molecular Design and Synthesis: We focused on the triphenyl-
phosphonium moiety to achieve mitochondrial localization and
the 2,6-dimethylnitrobenzene group as the NO-releasing moiety.
With the use of piperazine as a linker moiety, NBDNO is structurally
fixed in an extended form, which reduces of the possibility of intra-
molecular interaction between the NBD moiety and NO donor moi-
ety. The NO donor moiety was thus connected to a conjugated
containing 7.5% DMSO for 15 min in the presence of Fe–MGD, a
typical three-line spectrum of [(MGD)–Fe2+–NO] was observed in
ESR spectroscopy, indicating that NO was released from NBDNO
upon photoirradiation (Fig. 2). From a calibration curve obtained
by using separately prepared [(MGD)–Fe2+–NO], the rate of NO
release from NBDNO was found to be 10.8% for 15 min photoirra-
diation with the light-source (xenon lamp) of an irradiating appa-
ratus equipped with a UV filter (325–385 nm band-pass filter)
through a 50% ND filter (see Fig. S2 in Supplementary data).
Confocal microscopy: For confocal microscopy, HCT116 cells
were cultured in McCoy’s 5A culture medium. For the experiments,
cells were incubated for 2 days and then washed with D-PBS and
treated with 10 lM NBDNO for 30 min in the dark, followed by
washing once with D-PBS. The cells were then stained for 10 min
with MitoRedÒ, a well-established mitochondrial dye, and sub-
jected to confocal fluorescence microscopy. NBDNO was found to
be localized to mitochondria (Fig. 3), as expected from the presence
of the membrane-permeable and cationic triphenylphosphonium
moiety, because of the highly inside-negative membrane potential
across the inner mitochondrial membrane.
Intracellular release of NO from NBDNO: Photorelease of NO from
NBDNO in HCT116 cells was confirmed by using DAR-4M AM15, a
cell-membrane-permeable NO-specific fluorescence probe. Rhoda-
mine-based DAR-4M AM, which itself is nonfluorescent, is hydro-
lyzed intracellularly and the hydrolysis product reacts with NO
to form triazole DAR-4M T, which has strong red fluorescence in
p-electron system, which is known to be photoresponsive in biolog-
ical applications. NBDNO was synthesized as shown in Scheme 1
and characterized by 1H NMR, 13C NMR, HPLC, melting point mea-
surement, and mass spectroscopy.
Release of NO from NBDNO in vitro: NO release from NBDNO was
determined by means of an ESR spin-trapping method with Fe–N-
methyl-D-glucamine dithiocarbamate (Fe–MGD) complex, which is
known to react with NO to give a stable paramagnetic complex
[(MGD)–Fe2+–NO]. On ESR spectroscopy, the [(MGD)–Fe2+–NO]
complex gives a broadened three-line spectrum with aN = 1.25 mT
and giso = 2.04. When NBDNO was photoirradiated in MilliQ water
Scheme 1. Synthesis of NBDNO. Reagents and conditions: (a) Boc-piperazine, Cs2CO3, CHCl3, CH3CN; 4 N HCl/dioxane, CHCl3, quant; (b) DPPA, Et3N, t-BuOH, reflux 52.5%; 4 N
HCl/dioxane, CHCl3; NaHCO3 aq 64.0%; (c) SCCl2, Et3N, CH2Cl2, 77.3%; (d) Compound 2, Et3N, THF, DMF, 91.7%; (e) a,
a0-dibromoxylene, NaHCO3, DMF, 57.5% (f) PPh3, acetone,
CH2Cl2, 70.5%.

2002
T. Horinouchi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2000–2002
which serves to drive the localization of NBDNO to mitochondria,
and green-fluorescent NBD dye. NBDNO is expected be a useful
tool for studies on the biological roles of NO.
Acknowledgments
Figure 2. A representative ESR spectrum of a solution containing Fe–MGD and
NBDNO after photoirradiation (325–385 nm) via a 50% ND filter. Samples contained
750 lM NBDNO, 60 mM MGD, and 15 mM FeSO4 in MilliQ water containing 7.5%
This work was supported in part by Grants-in-Aid for Scientific
Research on Innovative Areas (Research in a Proposed Research
Area) (No. 21117514 to H.N.), Grants-in-Aid for Scientific Research
(No. 22590103 to H.N.) from the Ministry of Education, Culture,
Sports Science, and Technology, Japan, and JST PRESTO program
(H.N.), and the Sasagawa Scientific Research Grant from the Japan
Science Society (No. 22-339, T.H.).
DMSO. ESR spectra were recorded after photoirradiation for 15 min with
a
modulation width of 1.25 G and a microwave power of 10 mW.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.02.027.
References and notes
Figure 3. HCT116 human colon cancer cells were stained with MitoRedÒ and
NBDNO, and observed with a confocal fluorescence microscope. The distribution of
MitoRedÒ (left, red), the distribution of NBDNO (center, green), and the merged
image (right) of the same field are shown.
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