Visualization of nitroxyl in living cells by a chelated copper(II) coumarin complex

Article abstract of DOI:10.1021/ol103077q

The coumarin-based probe Cu(II)-COT1 was successfully developed for the detection of HNO on the basis of the reduction reaction. In addition, highly selective "turn on" type fluorogenic behavior upon the addition of Angeli's salt (Na₂N₂O₃) was also applied to bioimaging in A375 cells.

Full text of DOI:10.1021/ol103077q

ORGANIC
LETTERS
2011
Vol. 13, No. 6
1290–1293
Visualization of Nitroxyl in Living Cells by
a Chelated Copper(II) Coumarin Complex
Yi Zhou, Ke Liu, Ju-Ying Li, Yuan Fang, Tian-Chu Zhao, and Cheng Yao*
State Key Laboratory of Materials-Oriented Chemical Engineering and College of
Science, Nanjing University of Technology, Nanjing 210009, People’s Republic of China
yaochengnjut@126.com
Received December 20, 2010
ABSTRACT
The coumarin-based probe Cu(II)-COT1 was successfully developed for the detection of HNO on the basis of the reduction reaction. In addition,
highly selective “turn on” type fluorogenic behavior upon the addition of Angeli’s salt (Na2N2O3) was also applied to bioimaging in A375 cells.
Nitric oxide (NO) has received considerable attention
due to its role as active signal-inducing messenger biomo-
lecule in immune systems.1 NO at a moderate concentra-
tion plays a critical role in physiological processes such as
anticancer activity, vasodilation, synaptic activity, and
neurotransmission.2 Misregulation of NO production
has been associated with pathological conditions such as
cancer, ischemia, septic shock, inflammation, and neuro-
degeneration.3 One of the nitrogen oxides relevant to biol-
ogy is known as nitroxyl (HNO), which is the reduced/
protonated form of the signaling agent nitric oxide (NO).
HNO can be formed directly from nitric oxide synthase
under the appropriate conditions.4 Biologically, HNO also
increases cardiac contractility in both normal and failing
canine hearts through mechanisms that include increased
sarcoplasmic reticulum calcium release and uptake and
increased calcium dependent force development in cardiac
tissue.5 Thus, development of sensitive and selective meth-
ods for the detection of biological NO and HNO is of
importance.
There are many techniques available for detecting NO
and HNO, such as electrochemistry, colorimetry, electron
paramagnetic resonance (EPR), and chemiluminescence.6
However, these methods cannot be used to visualize NO
and HNO in vitro and in vivo. Fluorescence analysis has
high sensitivity and selectivity, and if it were to be
(5) (a) Paolocci, N.; Saavedra, W. F.; Miranda, K. M.; Martignani,
C.; Isoda, T.; Hare, J. M.; Espey, M. G.; Fukuto, J. M.; Feelisch, M.;
Wink, D. A.; Kass, D. A. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 10463–
10468. (b) Paolocci, N.; Katori, T.; St. Champion, H. C.; John, M. E.;
Miranda, K. M.; Fukuto, J. M.; Wink, D. A.; Kass, D. A. Proc. Natl.
Acad. Sci. U.S.A. 2003, 100, 5537–5542. (c) Dai, T. Y.; Tian, Y.;
Tocchetti, C. G.; Katori, T.; Murphy, A. M.; Kass, D. A.; Paolocci,
N.; Gao, W. D. J. Physiol. 2007, 580, 951–960. (d) Reisz, J. A.; Klorig,
E. B.; Wright, M. W.; King, S. B. Org. Lett. 2009, 11, 2719–2721.
(6) (a) Malinski, T.; Taha, Z. Nature 1992, 358, 676–678. (b) Ridnour,
L. A.; Sim, J. E.; Hayward, M. A.; Wink, D. A.; Martin, S. M.; Buettner,
G. R.; Spitz, D. R. Anal. Biochem. 2000, 281, 223–229. (c) Nagano, T.;
Yoshimura, T. Chem. Rev. 2002, 102, 1235–1269. (d) Hetrick, E. M.;
Schoenfisch, M. H. Annu. Rev. Anal. Chem. 2009, 2, 409–433.
(1) (a) Palmer, R. M. J.; Ferrige, A. G.; Moncada, S. Nature 1987,
327, 524–526. (b) Butler, A. R.; Williams, D. L. H. Chem. Soc. Rev. 1993,
22, 233–241. (c) Murad, F. Angew. Chem., Int. Ed. 1999, 38, 1856–1868.
(d) Furchgott, R. F. Angew. Chem., Int. Ed. 1999, 38, 1870–1880. (e)
Zimmet, J. M.; Hare., J. M. Circulation 2006, 114, 1531–1544.
(2) (a) Xu, W.; Liu, L. Z.; Loizidou, M.; Ahmed, M.; Charles, I. G.
Cell Res. 2002, 12, 311–320. (b) Hou, Y.; Wang, J.; Andreana, P. R.;
Cantauria, G.; Tarasia, S.; Sharp, L.; Braunschweiger, P. G.; Wang,
P. G. Bioorg. Med. Chem. Lett. 1999, 9, 2255–2256.
(3) (a) Tfelt-Hansen, J.; Ferreira, A.; Yano, S.; Kanuparthi, D.;
Romero, J. R.; Brown, E. M.; Chattopadhyay, N. Am. J. Physiol.
2005, 288, E1206–E1213. (b) Lala, P. K. Cancer Metastasis Rev. 1998,
17, 1–6. (c) Nelson, E. J.; Connolly, J.; McArthur, P. Biol. Cell 2003, 95,
3–8.
(4) (a) Fukuto, J. M.; Dutton, A. S.; Houk, K. N. ChemBioChem
2005, 6, 612–619. (b) Hobbs, A. J.; Fukuto, J. M.; Ignarro, L. J. Proc.
Natl. Acad. Sci. U.S.A. 1994, 91, 10992–10996.
(7) (a) Zheng, H.; Shang, G. Q.; Yang, S. Y.; Gao, X.; Xu, J. G. Org.
Lett. 2008, 10, 2357–2360. (b) Kojima, H.; Nakatsubo, N.; Kikuchi, K.;
Kawahara, S.; Kirino, Y.; Nagoshi, H.; Hirata, Y.; Nagano, T. Anal.
Chem. 1998, 70, 2446–2453. (c) Sasaki, E.; Kojima, H.; Nishimatsu, H.;
Urano, Y.; Kikuchi, K.; Hirata, Y.; Nagano, T. J. Am. Chem. Soc. 2005,
127, 3684–3685. (d) Gabe, Y.; Urano, Y.; Kikuchi, K.; Kojima, H.;
Nagano, T. J. Am. Chem. Soc. 2004, 126, 3357–3367. (e) Zhang, R.; Ye,
Z.; Wang, G.; Zhang, W.; Yuan, J. Chem. Eur. J. 2010, 16, 6884–6891.
r
10.1021/ol103077q
Published on Web 02/15/2011
2011 American Chemical Society

incorporated into a membrane-permeable probe, it could
be used to detect NO and HNO at the cellular level. A
number of groups have developed a variety of fluorescent
probes for NO and HNO, and these have been partially
successful for fluorescent imaging agents in living cells.7-9,12
However, these probes have some undesirable character-
istics related to their compatibility with living cells,
pH-dependent fluorescence, water solubility, and mem-
brane permeability.10,8b Membrane permeability is the key
feature for imaging experiments in intact cells for which
continual permeation of intracellular with artificial physio-
logical conditions is required.11 Fluorescein12 and boron-
dipyrromethene (BODIPY)9 dyes have been designed as
fluorophore platforms for the construction of efficient
membrane-permeable probes. Coumarin and its deriva-
tives are used extensively as fluorescence labeling reagents
for their excellent photophysical properties of high fluor-
escence quantum yield and efficient membrane permeabi-
lity.13 Therefore, we designed a probe for nitroxyl (HNO)
including a coumarin chromophore and a tripodal dipico-
lylamine receptor, which was attachedvia a triazole bridge.
The receptor provides a rigid Cu(II) binding site spacer
between the coumarin fluorophore and chelating ligand.
This type of probe was previously reported by Lippard’s
group, and the aim of this work was to produce a probe
with an improved response compared to their BODIPY
probe. With the aid of Lippard’s design strategy,9 the
probe also provides N bridges to minimize the distance
between the Cu2þ binding site and coumarin fluo-
rophore, which will ensure strong fluorescence quenching
in the off state of Cu(II) coumarin. Upon interaction with
HNO, chelated Cu(II) coumarin is reduced and the Cu(I)
coumarin complex forms, which induces an increase in the
fluorescence intensity. Changes in [HNO] under physiologi-
cal conditions will exhibit “turn-on” type fluorogenic beha-
vior, which can be detected by measuring the ratio of green
fluorescence intensity with good sensitivity and selectivity.
The synthesis of coumarin-trizole (COT1) and a pro-
posed mechanism are shown in Scheme 1. Diazotization of
Scheme 1. Synthesis of Coumarin-Trizole (COT1) and
Proposed Mechanism of Interaction with HNO To Generate
Fluorescence
amino-coumarin with NaN3 afforded azide-coumarin 3,
which was further installed by the copper(I)-mediated click
cycloaddition14 of alkyne 2 to give COT1 in 63% yield. The
chelated complex CuII[COT1]Cl2 was easily prepared by
addition of CuCl2 to COT1. This complex was soluble and
stable under physiological conditions in saline. COT1 and
Cu(I)-COT1 are both green emission fluorophores, and
they each displayed one absorption band in the visible
region centered at 400 nm (COT1, 8.08 ꢀ 104 M-1 cm-1
)
and 408 nm (Cu(I)-COT1, 8.15 ꢀ 104 M-1 cm-1) (Figure
S1, Supporting Information). Acid-base fluorescence titra-
tions revealed that the fluorescence intensity of Cu(I)-
COT1/Cu(II)-COT1 was unaffected by pH values between
6.08 and 9.41 (excitation at 415 nm). This suggests that the
probe would work well at physiological pH, although the
fluorescence intensity of Cu(I)-COT1 was quenched slightly
at pH <6.0 (Figures S2 and S3, Supporting Information).
The emission profile of COT1 showed typical coumarin
green fluorescence at 499 nm, with a high quantum yield
(Φf = 0.63) compared to that of 3 (Φf = 0.03), suggesting
the cycloaddition of 3 leads an increase in the electron-
donating ability in emission of coumarin. When 1 equiv of
Cu2þ was added to COT1 in aqueous solution, a dramatic
fluorescence quenching was observed (23.6-fold). This
could be attributed to the photoinduced electron transfer
(PET)15 from the coumarin fluorophore to the chelated
Cu2þ. The association constant of COT1 with Cu2þ was
determined to be 7.9 ꢀ 105 M-1 on the basis of the fluo-
rescence titration experiments (Figure S4; see the Support-
ing Information). The fluorescence intensity increased
(8) (a) Lim, M. H.; Lippard, S. J. Acc. Chem. Res. 2007, 40, 41–51. (b)
Hilderbrand, S. A.; Lim, M. H.; Lippard, S. J. J. Am. Chem. Soc. 2004,
126, 4972–4978. (c) Tonzetich, Z. J.; McQuade, L. E.; Lippard, S. J.
Inorg. Chem. 2010, 49, 6338–6348. (d) Marti, M. A.; Bari, S. E.; Estrin,
D. A.; Doctorovich, F. J. Am. Chem. Soc. 2005, 127, 4680–4684. (e)
Tennyson, A. G.; Do, L.; Smith, R. C.; Lippard, S. J. Polyhedron 2007,
26, 4625–4630.
(9) Rosenthal, J.; Lippard, S. J. J. Am. Chem. Soc. 2010, 132, 5536–
5537.
(10) (a) Smith, R. C.; Tennyson, A. G.; Lim, M. H.; Lippard, S. J.
Org. Lett. 2005, 7, 3573–3575. (b) Do, L.; Smith, R. C.; Tennyson, A. G.;
Lippard, S. J. Inorg. Chem. 2006, 45, 8998–9005.
(11) McQuadea, L. E.; Ma, J.; Lowe, G.; Ghatpande, A.; Gelperin,
A.; Lippard, S. J. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 8525–8530.
(12) (a) Lim, M. H.; Xu, D.; Lippard, S. J. Nat. Chem. Biol. 2006, 2,
375–380. (b) Lim, M. H.; Wong, B. A.; Pitcock, W. H., Jr.; Mokshagundam,
D.; Baik, M. H.; Lippard, S. J. J. Am. Chem. Soc. 2006, 128, 14364–
14373. (c) Pluth, M. D.; McQuade, L. E.; Lippard, S. J. Org. Lett. 2010,
12, 2318–2321.
(13) (a) Trenor, S. R.; Shultz, A. R.; Love, B. J.; Long, T. E. Chem.
Rev. 2004, 104, 3059–3077. (b) Lim, N. C.; Schuster, J. V.; Porto, M. C.;
Tanudra, M. A.; Yao, L.; Freake, H. C.; Bruckner, C. Inorg. Chem.
2005, 44, 2018–2030. (c) Ray, D.; Bharadwaj, P. K. Inorg. Chem. 2008,
47, 2252–2254. (d) Maity, D.; Govindaraju, T. Inorg. Chem. 2010, 49,
7229–7231. (e) Yuan, L.; Lin, W.; Song, J. Chem. Commun. 2010, 46,
7930–7932.
(14) (a) Boren, B. C.; Narayan, S.; Rasmussen, L. K.; Zhang, L.;
Zhao, H.; Lin, Z.; Jia, G.; Fokin, V. V. J. Am. Chem. Soc. 2008, 130,
8923–8930. (b) Sivakumar, K.; Xie, F.; Cash, B. M.; Long, S.; Barnhill,
H. N.; Wang, Q. Org. Lett. 2004, 6, 4603–4306.
(15) (a) Chen, X.; Nam, S. W.; Kim, G. H.; Song, N.; Jeong, Y.; Shin,
I.; Kim, S. K.; Kim, J.; Park, S.; Yoon, J. Chem. Commun. 2010, 46,
8953–8955. (b) Royzen, M.; Dai, Z.; Canary, J. W. J. Am. Chem. Soc.
2005, 127, 1612–1613.
Org. Lett., Vol. 13, No. 6, 2011
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Figure 1. Fluorescence titration of Cu(II)-COT1 (1 μM) in
HEPES buffer (50 mM, 0.1 M KNO3, pH 7.4) in the presence of
different amounts of Angeli’s salt. The fluorescence intensity
was measured 20 min after the addition of Na2N2O3 with
excitation at 415 nm. Inset: fluorescence intensity at 499 nm as a
function of Na2N2O3 concentration.
Figure 3. EPR spectra recorded at 298 K for 50 μM Cu(II)-
COT1 in aqueous ethanol (50 mM, pH 7.0, v/v, 50/50) (black
line) and with excess Na2N2O3 (red line).
Aryl-dipicolylamino (DPA) motifs have been used as
components of Zn2þ sensors because of their high affinity
for Zn2þ 16
Therefore, it was necessary to investigate the
.
effect of Zn2þ on the detection of HNO. The addition of
Na2N2O3 to a Cu(II)-COT1 solution with excess Zn2þ (50
μM) could induce minimal fluorescence intensity changes
in comparison with that obtained with HNO alone (Figure
S6, see the Supporting Information).
While Cu(II)-COT1 was highly sensitive to HNO, it was
important to investigate its selectivity with various ROS
and RNS species (Figure 2). The probe exhibited a 17.2-
fold increase with the ratio responses at 499 nm upon
interaction with HNO. However, a much weaker response
was observed with other biologically relevant ROS and
RNS species, including NO3-, ClO-, H2O2, ONOO-,
ROO•, and NO2-. With NO, a 3.2-fold increase in fluor-
escence intensity was observed, and this relative lack of
induced fluorescence response could be used to discrimi-
nate between NO and HNO. Competitive experiments
were conducted with 20 μM Na2N2O3 and various ROS
and RNS. The fluorescence intensity did not vary much in
comparison with that produced by HNO þ Cu(II)-COT1.
Furthermore, the response of the probe to HNO could be
detected simply by visual inspection (Figure 3). These
results indicate that Cu(II)-COT1 displays high selectivity
toward HNO over other ROS and RNS species.
Figure 2. Fluorescence responses of Cu(II)-COT1 (1 μM) to
various 10 μM ROS and RNS species (2 mM for saturated NO
aqueous solution). Bars represent the final (Ff) over the initial
(Fi) integrated emission at 499 nm. Spectra were acquired in
HEPES buffer (50 mM, 0.1 M KNO3, pH 7.4) solutions. The
gray bars represent the addition of the competing ROS or RNS
to a 1 μM solution of Cu(II)-COT1. The black bars represent the
change of the emission that occurred upon the subsequent
addition of 20 μM Na2N2O3 to the above solutions.
The EPR spectra of Cu(II)-COT1 in aqueous solution
showed typical paramagnetism17 with a symmetric line with
g = 2.10 (Figure 3). The introduction of excess Na2N2O3
to the Cu(II)-COT1 aqueous solution resulted in a rapid
decrease in the EPR signal because of the diamagnetism of
the reduced Cu(I)-COT1. The reduction of Cu2þ was
corroborated by the ESI-MS spectra of the probe and
steadily with increasing Na2N2O3 concentration (Figure 1)
until it reached a plateau at 20 μM Na2N2O3, which
corresponded to a 17.2-fold increase in fluoresence insten-
sity compared to the blank [HNO]. This indicates that
complete reduction of Cu(II)-COT1 occurred with 20 μM
Na2N2O3. The HNO induced a dramatic change at 499 nm
in the fluorescence intensity, which was much higher than
the result obtained with the early reported BODIPY-based
sensor.9 The fluorescence signal was gradually enhanced until
the equilibration showed a steady peak at 499 nm (∼8 min;
see Figure S5 in the Supporting Information), compared to
its analogues (which needed 20 min equilibrium time).7c
(16) (a) Qian, F.; Zhang, C.; Zhang, Y.; He, W.; Gao, X.; Hu, P.;
Guo, Z. J. Am. Chem. Soc. 2009, 131, 1460–1468. (b) Hanaoka, K.;
Kikuchi, K.; Kojima, H.; Urano, Y.; Nagano, T. J. Am. Chem. Soc.
2004, 126, 12470–12476.
(17) Neuman, N. I.; Perec, M.; Gonzalez, P. J.; Passeggi, M. C. G.;
Rizzi, A. C.; Brondino, C. D. J. Phys. Chem. A 2010, 114, 13069–13075.
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Org. Lett., Vol. 13, No. 6, 2011

A375 cells were first incubated with 5 μM Cu(II)-COT1
in phosphate-buffered saline for 30 min at 37 °C, which
produced very faint intracellular fluorescence (Figure
4b). Then, the treated cells were incubated with 50 μM
Na2N2O3 and the fluorescence signal was monitored at
regular intervals. The signal produced by these cells
increased over time (Figure 4c-e). The major fluores-
cence intensity in the cells was localized in the peri-
nuclear space, which suggests that the NO species did
not reach the nucleolus. The overlay of fluorescence
and bright-field images indicated that Cu(II)-COT1
had a good cell membrane permeability. Furthermore,
we employed the MTT assay to investigate the cyto-
toxicity of 5 μM Cu(II)-COT1 after 12 h of incubation
with A375 cells (see theSupporting Information). The
Cu(II)-COT1 probe had low toxicity with the A375
cells, and the tests showed 82.1% cell viability. This low
toxicity is important for HNO biological imaging
studies.
In summary, a Cu(II) coumarin conjugate probe was
developed that acted as a dual-response probe to HNO for
both fluorescence and EPR detection. The probe was
HNO specific and provided high selectivity over other
ROS and RNS species. Great changes in the fluorescence
color could be distinguished by the naked eye. Fluores-
cence imaging shows that Cu(II)-COT1 could be used for
detection of changes in HNO levels in living cells with low
cytotoxicity. The design strategy and remarkable photo-
physical properties of the coumarin green fluorescent
moieties will be useful for the development of fluorescent
probes for ROS and RNS.
Figure 4. Fluorescence images of A375 cells: (a) bright-field
image of cells shown in panel; (b) cells incubated with 5 μM
Cu(II)-COT1 in PBS buffer (50 mM, pH 7.2) for 30 min; (c-e)
the cells in (b) incubated with Na2N2O3 (50 μM) for 1, 6, and 15,
respectively; (f) overlay image of (a) and (e).
Cu(I)-COT1. Two major peaks at m/z 558.2 and 616.3
(calcd 558.2 and 616.1) corresponding to [COT1 þ Cu(I)]þ
and [COT1 þ Cu(I) þ Na þ Cl]þ were observed when
5 μM Na2N2O3 was added. By comparison, the probe
without HNO only exhibited a peak at m/z 593.2 (calcd
593.1), which corresponded to [COT1 þ Cu(II) þ Cl]þ. In
addition, submillimolar cysteine18 and sodium ascorbate19
could also be used to restore the typical coumarin green
fluorescence (see Figure S7 in the Supporting Information)
because of the reduction of chelated Cu(II)-coumarin. A
peak for this reduction product was observed in the ESI-
MS spectra at m/z 558.2 (calcd 558.2), which corresponded
to the cationic [COT1 þ Cu(I)]þ complex.
Acknowledgment. This work was supported by the Open
Fund of the State Key Laboratory of Materials-Oriented
Chemical Engineering (No. KL09-9), the postgraduate
practice innovation fund of Jiangsu province, and the
doctoral thesis innovation fund of the Nanjing University
of Technology.
The probe was then applied to fluorescence imaging
of HNO in living cells. Human malignant melanoma
(18) A reviewer suggested that we investigate the fluorescence in-
tensity change of Cu(II)-COT1 in the presence of other thiol-containing
amino acids; however, we considered that other thiol-containing amino
acids have no reduction properties and this work deals with a probe for
HNO. Therefore, there is no need to investigate other thiol-containing
amino acids.
Supporting Information Available. Text, figures, and a
table giving synthesis and experimental details and addi-
tional spectroscopic data. This material is available free
of charge via the Internet at http://pubs.acs.org.
(19) Taki, M.; Iyoshi, S.; Ojida, A.; Hamachi, I.; Yamamoto., Y. J. Am.
Chem. Soc. 2010, 132, 5938–5939.
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