Visible light-induced nitric oxide release from a novel nitrobenzene derivative cross-conjugated with a coumarin fluorophore

Article abstract of DOI:10.1016/j.bmcl.2014.10.075

Nitric oxide (NO) is a well-known free-radical molecule which is endogenously biosynthesised and shows various functions in mammals. To investigate NO functions, photocontrollable NO donors, compounds which release NO in response to light, are expected to

Full text of DOI:10.1016/j.bmcl.2014.10.075

Bioorganic & Medicinal Chemistry Letters 24 (2014) 5660–5662
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Visible light-induced nitric oxide release from a novel nitrobenzene
derivative cross-conjugated with a coumarin fluorophore
Kai Kitamura a, Naoya Ieda a, Kazuhiro Hishikawa a, Takayoshi Suzuki b, Naoki Miyata a,
Kiyoshi Fukuhara c, Hidehiko Nakagawa a,
⇑
a Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi 467-8603, Japan
b Kyoto Prefectural University of Medicine, 13, Taishogun, Nishitakatsukasa-cho, Kita-ku, Kyoto 403-8334, Japan
c School of Pharmacy, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Nitric oxide (NO) is a well-known free-radical molecule which is endogenously biosynthesised and shows
various functions in mammals. To investigate NO functions, photocontrollable NO donors, compounds
which release NO in response to light, are expected to be potentially useful. However, most of the
conventional NO donors require harmful ultra-violet light for NO release. In this study, two dimethylni-
trobenzene derivatives conjugated with coumarins were designed, synthesized and evaluated as
photocontrollable NO donors. The optical properties and efficiency of photo-induced NO release were
dependent upon the nature of the conjugation system. One of these compounds, Bhc-DNB (1), showed
spatiotemporally well-controlled NO release in cultured cells upon exposure to light in the less-cytotoxic
visible wavelength range (400–430 nm).
Received 30 August 2014
Revised 21 October 2014
Accepted 23 October 2014
Available online 29 October 2014
Keywords:
Nitric oxide
NO donor
Visible light-control
Caged compound
Cross-conjugation
Ó 2014 Elsevier Ltd. All rights reserved.
Nitric oxide (NO) is a small-molecular, gaseous mediator that
plays important roles in various physiological processes, including
vasorelaxation,1 neuromodulation2 and biodefence.3 It may also be
involved in the pathophysiology of diseases such as cancer,4 Alz-
heimer’s disease5 and schizophrenia.6 Therefore, methods for pre-
cisely controlled release of NO are required for research purposes
and might ultimately be clinically relevant. Since NO is unstable
under ambient conditions, compounds that release NO in situ,
NO donors, have been developed and employed for NO research.7
Since physiological NO production by nitric oxide synthase (NOS)
is precisely regulated by enzymes and signal transduction machin-
ery, photocontrolled release of NO from a donor molecule seems
particularly attractive as a means to achieve spatiotemporally
controlled intracellular NO release.
We previously reported on photo-induced NO release from 6-
nitrobenzo[a]pyrene (6-nitroBaP),8 and based on this finding, we
developed 2,6-dimethylnitrobenzene (2,6-DNB) derivatives as
photocontrollable NO donors.9 These 2,6-DNB derivatives have an
aromatic nitro group that is not planar with respect to the benzene
ring due to the steric effect of the two methyl groups at ortho-posi-
tions and this twisted conformation is considered to facilitate
photo-induced isomerisation of the nitro group to nitrite ester,
which subsequently undergoes homolytic fission to release NO
(Fig. 1). As photocontrollable NO donors, these 2,6-DNB derivatives
are attractive, because they are stable (easy to handle), not metal-
containing (non-cytotoxic) and, above all, unique in their mecha-
nism of NO release. However, they have a serious limitation, in that
their maximum absorption band for NO release is mainly in the
UV-A range, which is harmful to living cells. To develop novel NO
donors suitable for cellular applications, we focused on coumarins
as compact fluorophores, particularly those with an electron-
donating group at the 7-position, such as 7-hydroxy- and 7-amino-
coumarins. The absorption of such coumarin derivatives is
expected to be shifted to longer wavelength through intermolecu-
lar charge transfer (ICT). Therefore, we designed and synthesized
two novel 2,6-DNB derivatives conjugated with coumarin fluoro-
phores, that is, Bhc-DNB (1) and DEAMC-DNB (2) (Fig. 2). In
Bhc-DNB (1), the olefin linker to the 2,6-DNB moiety is cross-con-
jugated10 with 6-bromo-7-hydroxycoumarin (Bhc)11 at the 4-posi-
tion of the coumarin ring, while in DEAMC-DNB (2), the linker is
linearly conjugated with 7-(diethylamino)-4-methylcoumarin at
the 3-position. In the latter case, the nitro group of 2,6-DNB is
expected to be an efficient electron acceptor for ICT, whereas this
is not in the case in Bhc-DNB (1). Thus, the electron density of
the NO-releasing nitro group of Bhc-DNB (1) in the photo-excited
state is expected to be lower than that of DEAMC-DNB (2).
These molecules were synthesized as shown in Scheme S1 (see
Supplementary material). Their chemical structure and purity were
⇑
Corresponding author.
http://dx.doi.org/10.1016/j.bmcl.2014.10.075
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

K. Kitamura et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5660–5662
5661
NO–Fe2+–MGD2 complex showing typical triplet signals at around
330 mT in 1 GHz ESR spectroscopy. Aqueous solutions (50% DMSO,
2.5 mM potassium phosphate buffer, pH 7.35) of the compounds in
the presence of 1.5 mM Fe2+–MGD2 complex were photoirradiated
under the indicated conditions, and the solutions were subjected to
ESR analysis. As shown in Figure 4, solutions of Bhc-DNB (1) irra-
diated at 325–385 nm (UV-A light) or 400–430 nm (violet light)
showed a typical triplet signal assigned to NO–Fe2+–MGD2 com-
plex in the ESR spectrum, confirming NO release from the com-
pound (Fig. 4A and B). In contrast, a solution of DEAMC-DNB (2)
irradiated with 430–460 nm light did not show the triplet signal
in the ESR spectrum, which means that NO is not released by phot-
oirradiation in this wavelength range (Fig. 4C). Thus, we found that
Bhc-DNB (1) released NO upon light irradiation around its absorp-
tion band, whereas DEAMC-DNB (2) did not. This interesting result
presumably reflects the difference in conjugation systems, namely
the difference of electron density of the NO-releasing nitro group
in the excited state. It appears that ICT in the DEAMC-DNB (2) mol-
ecule gives a double-bond character to the CAN bond of the NO-
releasing nitro group, which prevents isomerisation of the nitro
group to nitrite ester because of the increase of its planarity
(Fig. S2, Supplementary material). In order to confirm the expecta-
tion, the nature of the nitro group was examined by infrared spec-
troscopy, which showed that the property of the nitro group of
DEAMC-DNB (2) is similar to that of nitrobenzene, which has no
steric effect of methyl groups, than that of 2,6-DNB such as 2-
nitromesitylene and Bhc-DNB (1). (Fig. S3 and Table S1, Supple-
mentary material) The observation that Bhc-DNB (1) could release
NO in response to visible light suggested that it is potentially avail-
able as an NO donor controllable with visible light.
Figure 1. Proposed mechanism of photo-induced NO release from 2,6-dimethyl-
nitrobenzene (2,6-DNB) derivatives.
Figure 2. Chemical structures of Bhc-DNB (1) and DEAMC-DNB (2).
Next, we investigated the photo-decomposition products of
Bhc-DNB (1) by LC/ESI–MS. It was expected that one of the
photo-decomposition products of Bhc-DNB (1) might be a dimeth-
ylphenol compound (17) whose phenol group is derived from the
nitro group of Bhc-DNB (1), so we attempted to detect 17 by single
ion monitoring at m/z 389, a mono-isotopic mass derived from the
compound containing 81Br. An aqueous solution (50% DMSO,
20 mM potassium phosphate buffer, pH 7.35) of 100 lM Bhc-
Figure 3. UV–visible absorption spectra of Bhc-DNB (1) and DEAMC-DNB (2); e360
of Bhc-DNB (1) is 11217 MÀ1 cmÀ1
,
e426
(kmax) of DEAMC-DNB (2) =
nm
nm
DNB (1) was irradiated with 325–385 nm (UV-A) light for 15 min
and the resulting solution was subjected to LC/ESI–MS analysis.
The retention time of a major photo-decomposition product in
the HPLC chromatogram corresponded to that of the peak detected
by single ion monitoring (m/z 389) (Fig. S4, Supplementary mate-
rial). Moreover, the ESI–MS spectrum of this peak showed m/z
387, confirming the detection of 17 (Fig. S5, Supplementary mate-
rial). Thus, Bhc-DNB (1) is mainly photolyzed to 17 which is a
one-electron reduced form of the phenoxyl radical generated by
photo-induced NO release.
47,345MÀ1 cmÀ1
.
confirmed by 1H NMR, 13C NMR, mass spectrometry and elemental
analysis.
First of all, we measured the ultraviolet-visible absorption spec-
tra to examine the effect of the difference in conjugation system.
As we had expected, DEAMC-DNB (2) showed a quite elongated
absorption band at around 400–500 nm owing to efficient ICT,
whereas Bhc-DNB (1) showed an absorption band at around
350–430 nm, which corresponds to that of Bhc itself.11 In other
words, the absorption band of Bhc-DNB (1) was not shifted, pre-
sumably due to the cross-conjugated structure (Fig. 3).
Next, to confirm NO release from the compounds we adopted an
ESR spin trapping method with ferrous N-methylglucamine dithio-
carbamate (Fe2+–MGD2) complex, which traps NO to yield an
We next evaluated the decomposition quantum yield (
U),
which is parameter of photo-decomposition efficiency, of
a
Bhc-DNB (1) from the decrement of the HPLC peak area after phot-
oirradiation and the photon quantity of the light source measured
by utilizing potassium ferrioxalate photo-reduction, whose
quantum yield has been reported.11 It was found that the
Figure 4. ESR spectra of aqueous solutions (50% DMSO, 2.5 mM potassium phosphate buffer, pH 7.35) containing the test compounds and 1.5 mM Fe2+–MGD2 complex after
photo-irradiation. (A) 100
M Bhc-DNB (1), 325–385 nm (Xe lamp, 15 mW/cm2 at 360 nm, 10 min); (B) 500 M Bhc-DNB (1), 400–430 nm (Xe lamp, 190 mW/cm2 at
415 nm, 15 min); (C) 100
M DEAMC-DNB (2), 430–460 nm (Xe lamp, 180 mW/cm2 at 445 nm, 15 min). Control (blank) data are shown in Figure S1, Supplementary material.
l
l
l

5662
K. Kitamura et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5660–5662
Figure 5. Fluorescence detection images of NO released from Bhc-DNB (1) in HCT116 cells. Cultured HCT116 cells were treated with 25 lM Bhc-DNB (1) and 10 lM DAR-4 M
AM. Before and after photoirradiation, the cells were observed with a fluorescence microscope. (A) is a differential interference contrast (DIC) image of (B) and (C); (B) is the
fluorescence image before photoirradiation; (C) is the fluorescence image after photoirradiation (330–380 nm, 10 min) within the irradiation circle indicated in (B); (D) and
(F) are DIC images of (E) and (G), respectively; (E) is the fluorescence image before photoirradiation; (G) is the fluorescence image after photoirradiation (400–430 nm,
15 min) observed in the same the dish as that used for (E). (The control is shown in Fig. S6, Supplementary material.)
decomposition quantum yield at 358 nm
(
U358
)
nm
was
Supplementary data
0.053 0.04, which is comparable with that of other photo-caged
compounds, including some compounds bearing an o-nitrobenzyl
group,12 a well-known photo-removable protecting group, and
Bhc derivatives.13
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.10.
075.
Finally, we applied Bhc-DNB (1) to living cells and also explored
its cellular localization. To evaluate the NO-releasing ability of
Bhc-DNB (1) in cells, we adopted DAR-4M AM, which is a cell-per-
meable red fluorescent probe for NO developed by Nagano et al.14
HCT116 human colon cancer cells were loaded with Bhc-DNB (1)
and the probe. After UV-A (330–380 nm) light irradiation of cells
within the indicated irradiation circle under microscope observa-
tion, the cells were observed by means of fluorescence microscopy.
As shown in Figure 5, red fluorescence of the NO probe was
observed in the photoirradiated area. Furthermore after visible-
light (400–430 nm) irradiation of the whole cell culture dish, an
increase of red fluorescence was also observed, while no such
increase was observed in cells without Bhc-DNB (1) (Fig. S6, Sup-
plementary material). The compound appeared to be distributed
in cytoplasm. These results confirmed that Bhc-DNB (1) is a novel
NO donor which is cell-applicable and controllable with visible
light (Fig. S6, Supplementary material).
In conclusion, we designed and synthesized two dimethylnitro-
benzene derivatives conjugated with coumarin fluorophores, Bhc-
DNB (1) and DEAMC-DNB (2). Bhc-DNB (1) released NO in
response to not only UV-A, but also visible light (400–430 nm),
while DEAMC-DNB (2) did not release NO in response to irradia-
tion at the wavelength of the absorption maximum. These results
are considered to reflect the difference in their conjugation sys-
tems. Thus, Bhc-DNB (1), a cross-conjugated compound, is avail-
able as an NO donor from which NO release is controllable with
visible light in living cells. The major photo-decomposition product
of Bhc-DNB (1) is a dimethylphenol compound (17). Bhc-DNB (1)
is expected to be superior to existing photocontrollable NO donors
for cellular studies that require precise, photocontrolled intracellu-
lar NO release, because release can be achieved with light in the
non-cytotoxic visible wavelength range.
References and notes
1. Ignarro, L. J.; Buga, G. M.; Wood, K. S.; Byrns, R. E.; Chaudhuri, G. Proc. Natl.
Acad. Sci. U.S.A. 1987, 84, 9265.
2. Sattler, R.; Xiong, Z.; Lu, W.-Y.; Hafner, M.; MacDonald, J. F.; Tymianski, M.
Science 1999, 284, 1845.
3. Wei, X. Q.; Charles, I. G.; Smith, A.; Ure, J.; Feng, G. J.; Huang, F. P.; Xu, D.;
Muller, W.; Moncada, S.; Liew, F. Y. Nature 1995, 375, 408.
4. Cobbs, C. S.; Brenman, J. E.; Aldape, K. D.; Bredt, D. S.; Israel, M. A. Cancer Res.
1995, 55, 727.
5. (a) Kuiper, M. A.; Visser, J. J.; Bergmans, P. L.; Scheltens, P.; Wolters, E. C. J.
Neurosci. 1994, 124, 46; (b) Cho, D. H.; Nakamura, T.; Fang, J.; Cieplak, P.;
Godzik, A.; Gu, Z.; Lipton, S. A. Science 2009, 324, 102.
6. Bernstein, H. G.; Keilhoff, G.; Steiner, J.; Dobrowolny, H.; Bogerts, B. CNS Neurol.
Disord.: Drug Targets 2011, 10, 792.
7. Wang, P. G.; Xian, M.; Tang, X.; Wu, X.; Wen, Z.; Cai, T.; Janczuk, A. J. Chem. Rev.
2002, 102, 1091.
8. Fukuhara, K.; Kurihara, M.; Miyata, N. J. Am. Chem. Soc. 2001, 123, 8662.
9. (a) Suzuki, T.; Nagae, O.; Kato, Y.; Nakagawa, H.; Fukuhara, K.; Miyata, N. J. Am.
Chem. Soc. 2005, 127, 11720; (b) Hishikawa, K.; Nakagawa, H.; Furuta, T.;
Fukuhara, K.; Tsumoto, H.; Suzuki, T.; Miyata, N. J. Am. Chem. Soc. 2009, 131,
7488; (c) Hishikawa, K.; Nakagawa, H.; Furuta, T.; Fukuhara, K.; Tsumoto, H.;
Suzuki, T.; Miyata, N. Bioorg. Med. Chem. Lett. 2010, 20, 302; (d) Horinouchi, T.;
Nakagawa, H.; Suzuki, T.; Fukuhara, K.; Miyata, N. Bioorg. Med. Chem. Lett. 2011,
21, 2000; (e) Horinouchi, T.; Nakagawa, H.; Suzuki, T.; Fukuhara, K.; Miyata, N.
Chem. Eur. J. 2011, 17, 4809.
10. (a) Valkenier, H.; Guédon, C. M.; Markussen, T.; Thygesen, K. S.; van der Molen,
S. J.; Hummelen, J. C. Phys. Chem. Chem. Phys. 2013, 16, 653; (b) Leu, W. C.; Fritz,
A. E.; Digianantonio, K. M.; Hartley, C. S. J. Org. Chem. 2012, 77, 2285.
11. Furuta, T.; Wang, S. S.; Dantzker, J. L.; Dore, T. M.; Bybee, W. J.; Callaway, E. M.;
Denk, W.; Tsien, R. Y. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 1193.
12. (a) Aujard, L.; Benbrahim, C.; Gouget, M.; Ruel, O.; Baudin, J. B.; Neveu, P.;
Jullien, L. Chem. Eur. J. 2006, 12, 6865; (b) Hasan, A.; Stengele, K.-P.; Giegrich,
H.; Cornwell, P.; Isham, K. R.; Sachleben, R. A.; Pfleiderer, W.; Foote, R. S.
Tetrahedron 1997, 53, 4247.
13. (a) Furuta, T.; Takeuchi, H.; Isozaki, M.; Takahashi, Y.; Kanehara, M.; Sugimoto,
M.; Watanabe, T.; Noguchi, K.; Dore, T. M.; Kurahashi, T.; Iwamura, M.; Tsien, R.
Y. ChemBioChem 2004, 5, 1119; (b) Bourbon, P.; Peng, Q.; Ferraudi, G.;
Stauffacher, C.; Wiest, O.; Helquist, P. Bioorg. Med. Chem. Lett. 2013, 25, 2162.
14. Kojima, H.; Hirotani, M.; Nakatsubo, N.; Kikuchi, K.; Urano, Y.; Higuchi, T.;
Hirata, Y.; Nagano, T. Anal. Chem. 2001, 73, 1967.
Acknowledgments
This work was supported by the JST PRESTO program (H.N.)
from Japan Science and Technology Agency, as well as by a JSPS
KAKENHI Grant Number 25293028 (H.N.).