A small-molecule two-photon probe for nitric oxide in living tissues

Article abstract of DOI:10.1002/chem.201201197

Two-photon microscopy (TPM) has become an indispensable tool in the study of biology and medicine due to the capability of this method for molecular imaging deep inside intact tissues. For the maximum utilization of TPM, a variety of two-photon (TP) probes for specific applications are needed. In this article, we report a small-molecule TP probe (ANO1) for nitric oxide (NO) that shows a rapid and specific NO response, a 68-fold fluorescence enhancement in response to NO, and a maximum TP-action cross-section of 170-GM (GM: 10 ^-50-cm⁴ photon^-1) upon reaction with excess NO. This probe can be easily loaded into cells and tissues and can real-time monitor NO in living tissues at 100-180-μm depth for longer than 1200-s through the use of TPM, with minimum interference from other biologically relevant species. Copyright

Full text of DOI:10.1002/chem.201201197

FULL PAPER
DOI: 10.1002/chem.201201197
A Small-Molecule Two-Photon Probe for Nitric Oxide in Living Tissues
Eun Won Seo,[a] Ji Hee Han,[b] Cheol Ho Heo,[a] Jae Ho Shin,[c]
Hwan Myung Kim,*[a] and Bong Rae Cho*[b]
Abstract:
Two-photon
microscopy
(ANO1) for nitric oxide (NO) that
shows a rapid and specific NO re-
sponse, a 68-fold fluorescence enhance-
ment in response to NO, and a maxi-
mum TP-action cross-section of
170 GM
(GM: 10ꢀ50 cm4 photonꢀ1)
(TPM) has become an indispensable
tool in the study of biology and medi-
cine due to the capability of this
method for molecular imaging deep
inside intact tissues. For the maximum
utilization of TPM, a variety of two-
photon (TP) probes for specific appli-
cations are needed. In this article, we
upon reaction with excess NO. This
probe can be easily loaded into cells
and tissues and can real-time monitor
NO in living tissues at 100–180 mm
depth for longer than 1200 s through
the use of TPM, with minimum inter-
ference from other biologically rele-
vant species.
Keywords: fluorescent probes · liv-
ing tissues · nitrogen oxides · two-
photon microscopy
probe
· two-photon
report
a
small-molecule TP probe
Introduction
the o-diaminobenzene moiety in these probes is converted
into triazine by reaction with the autooxidation products of
NO such as NO+ and N2O3, thereby blocking the photoin-
duced electron transfer (PeT) and allowing strong emis-
sion.[11–14] Lippardꢀs group reported a CuII–fluorescein-based
probe (CuFL) with a much faster response, which was
useful for real-time imaging of NO in live cells.[15–17] More
recently, a reaction-based turn-on probe has been developed
to detect NO in live cells.[18] These small-molecule probes do
not require transfection, unlike their protein counter-
parts.[19–21] However, the use of these probes with one-
photon microscopy (OPM) requires rather short excitation
wavelengths (<500 nm), which limits their application in
tissue imaging due to the shallow penetration depth
(<80 mm). Nagano and co-workers reported a cyanine-de-
rived NO probe for near-infrared (NIR) microscopy imag-
ing.[14] Although NIR microscopy is useful for imaging big
objects such as a whole mouse, it is not suitable for imaging
microlevel objects, such as cells and tissue slices, with high
resolution. Two-photon microscopy (TPM) overcomes these
problems. TPM, which employs two near-infrared photons
as the excitation source, offers a number of advantages over
one-photon microscopy, including increased depth of pene-
tration (>500 mm), localized excitation, and prolonged ob-
servation times.[22,23] In this context, TPM has become indis-
pensable to the study of biology and medicine due to its ca-
pability for molecular imaging deep inside intact tissues with
minimum interference from background emissions and mini-
mum photodamage.[24–26] However, to the best of our know-
Small-molecule-derived fluorescent probes are essential
tools for studying cell biology such as small-molecule signal-
ing.[1–5] Nitric oxide (NO) is a ubiquitous signaling molecule
that modulates a variety of physiological and pathological
processes in living organisms. NO plays a key role in synap-
tic activity in the brain and defends immune systems. On the
other hand, misregulation of NO production can cause vari-
ous diseases, such as cancer and neurodegenerative disor-
ders.[6–10] In order to understand its roles in biology, it is cru-
cial to monitor NO in intact tissues.
To detect NO in live cells, a number of fluorescent probes
derived from o-diaminobenzene have been developed, with
diaminofluoresceins such as 4,5-diaminofluorescein (DAF-2)
being commercially available.[11–14] It is well accepted that
[a] E. W. Seo,+ C. H. Heo, Prof. Dr. H. M. Kim
Division of Energy Systems Research
Ajou University
Suwon, 443-749 (Korea)
Fax: (+82)31-219-1615
E-mail: kimhm@ajou.ac.kr
[b] J. H. Han,+ Prof. Dr. B. R. Cho
Department of Chemistry
Korea University
1-Anamdong, Seoul, 136-701 (Korea)
Fax : (+82)2-3290-3544
E-mail: chobr@korea.ac.kr
[c] Prof. Dr. J. H. Shin
Department of Chemistry
Kwangwoon University
Seoul, 139-701 (Korea)
AHCTUNGTREGlNNNU edge, there has been no report of a two-photon (TP) probe
for NO that is applicable for deep-tissue imaging. We there-
fore designed a simple and efficient TP turn-on probe
(ANO1, Scheme 1) by considering the following require-
ments: 1) a low molecular weight for cell permeability,[27]
2) an appreciable water solubility to stain the cells and tis-
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201201197.
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Scheme 1. Synthesis of ANO1 and ANO1-T: a) DCC, HOBt, CH2Cl2,
b) SnCl2·2H2O, CH2Cl2. DCC: 1,3-dicyclohexylcarbodiimide; HOBt:
1-hydroxy-1H-benzotriazole.
sues,[28] 3) a high selectivity for NO, 4) a large turn-on re-
sponse for superior spatial resolution,[29] 5) a significant TP
cross-section for a bright TPM image, and 6) high photosta-
bility.
In this article, we present the design, synthesis, spectro-
scopic properties in both one- and two-photon mode, and
TPM-imaging application of ANO1, which can detect nitric
oxide in live cells and intact tissues for a long period of time
through the use of TPM.
Figure 1. a) One-photon absorption spectra of ANO1 (2 mm) after addi-
tion of 100 mm NO in PBS (pH 7.4). Spectra were acquired in PBS
(pH 7.4) at 0 and 15 min after addition of NO. b) One-photon fluores-
cence response with time for the reaction of ANO1 (2 mm) with NO
(100 mm) at 378C. Spectra were acquired in PBS (pH 7.4) between 0 and
15 min after addition of NO. Excitation at l=370 nm.
Results and Discussion
ANO1 exhibits an absorption maximum at labs =370 nm (ex-
tinction coefficient: e=12700mꢀ1 cmꢀ1) and a fluorescence
maximum at lfl =502 nm (fluorescence quantum yield: F=
0.009). The emission spectra of ANO1 showed gradual red-
shifts with increasing solvent polarity in the order 1,4-diox-
ane<N,N-dimethylformamide (DMF)<EtOH< buffer
(Figure S3 and Table S1 in the Supporting Information). The
large redshifts (>85 nm) with increasing solvent polarity in-
dicate the utility of ANO1 as an environment-sensitive
probe.
When excess NO was added to ANO1 in PBS, the fluores-
cence intensity (FEF=Fmax/Fmin) increased 68-fold within
5 min without affecting the absorption spectra, presumably
due to blocking of the PeT upon oxidation (Figure 1). The
large turn-on response is consistent with a 78-fold higher flu-
orescence quantum efficiency (F=0.70, Table 1) of ANO1-
T than that of ANO1. Moreover, the plot of the fluores-
cence intensity against the NO concentration was linear in
the range from 5 nm to 20 mm (Figure S4 in the Supporting
Information), which indicates that ANO1 can detect NO at
concentrations as low as 5 nm.
It has been well established that the combination of a recep-
tor for the target species and a 2-acetyl-6-dialkylaminonaph-
thalene (acedan) as the reporter with a glycinamide linkage
is a simple and effective strategy to design a TP turn-on
probe.[30–32] We have now developed an efficient TP probe
for NO based on an acedan as the reporter and an o-diami-
nobenzene as the reaction site for NO, linked by prolina-
mide (ANO1, Scheme 1), with the expectation that the TP
excited-fluorescence (TPEF) intensity would increase dra-
matically upon reaction with NO.[29] The acedan was adopt-
ed from our earlier work[30,31] and the o-diaminobenzene
from the work of Nagano and co-workers.[12–14] ANO1 was
prepared in 35% yield by the coupling of B and 2-nitro-1,4-
phenylenediamine followed by reduction (Scheme 1 and see
the Experimental Section). The reaction between ANO1
and NO, as monitored by fluorescence emissions (Figure 1b)
and LC-MS (Figure S1 in the Supporting Information), pro-
duced ANO1-T as the only major product.
The solubility of ANO1 and ANO1-T in phosphate-buf-
fered saline (PBS; 30 mm, containing 100 mm KCl, pH 7.4)
as determined by the fluorescence method is 4 mm,[28] which
is sufficient to stain cells (Figure S2 in the Supporting Infor-
mation). Under these simulated physiological conditions,
ANO1 showed high selectivity for NO over competing re-
active nitrogen and oxygen species (RNS, ROS), including
ꢀ
ꢀ
nitroxyl (HNO), nitrite (NO2 ), nitrate (NO3 ), hydrogen
peroxide (H2O2), peroxynitrite (ONOOꢀ), tert-butylhydro-
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Two-Photon Probe for Nitric Oxide in Living Tissues
FULL PAPER
Table 1. Photophysical data for compounds A, B, ANO1, and ANO1-T.[a]
due to the different structures of ANO1 and DAF-2.[35]
Moreover, the fluorescence response decreased significantly
under deoxygenated conditions, thereby confirming that the
oxidized form of NO is responsible for the detection (Fig-
ure S5 in the Supporting Information).[11–14] Furthermore,
ANO1 and ANO1-T are pH insensitive in the biologically
relevant pH range (Figure 2b). The combined results reveal
that ANO1 can detect NO under physiological conditions
with minimum interference from other RNS, ROS, AA, and
DHA, as well as from the pH value.
We then evaluated the ability of ANO1 to detect NO in a
two-photon mode. The TP-action cross-section was deter-
mined by investigating the TPEF spectra of ANO1 and
ANO1-T with rhodamine-6G as the reference (see the Ex-
perimental Section). Whereas the peak TP-action cross-sec-
tion (Fdmax) of ANO1 in PBS was too small to measure ac-
curately, that of ANO1-T was 170 GM at 750 nm under the
ð1Þ
fl
max
[nm][c]
ð2Þ
l
(10ꢀ4 e)
l
F[d]
l
Fdmax
[GM][f]
max
max
[nm][b]
[nm][e]
A
B
380 (1.24)
385 (1.44)
370 (1.27)
370 (1.22)
515
520
502
502
0.34
0.55
0.009
0.70
750
750
750
750
55
100
n.d.[g]
170
ANO1
ANO1-T
[a] All measurements were performed in PBS. [b] lmax value of the one-
photon absorption spectra. The numbers in parentheses are the molar ex-
tinction coefficients in mꢀ1 cmꢀ1. [c] lmax value of the one-photon emission
spectra. [d] Fluorescence quantum yield. [e] lmax value of the two-photon
excitation spectra. [f] The peak two-photon-action cross-section (GM:
10ꢀ50 cm4 photonꢀ1). [g] n.d.: not determined. The TPEF intensity was too
weak to measure the cross-section accurately.
peroxide (TBHP), hypochlorite (OClꢀ), superoxide (O2 ),
ꢀ
1
C
singlet oxygen ( O2), hydroxyl radical ( OH), and tert-butoxy
C
radical ( OtBu), as well as ascorbic acid (AA) and dehy-
droascorbic acid (DHA) (Figure 2a). The high selectivity
over AA and DHA is particularly interesting for biological
applications because DAF-2 does not show such selectivi-
ty.[33,34] It is well known that the fluorophore part can affect
the binding affinity of the probe, so this outcome might be
same
conditions
(Table 1
and
Figure 3;
GM:
Figure 3. Two-photon-action spectra of 1 mm A, B, and ANO1-T in PBS
(pH 7.4). The estimated uncertainties for the TP-action cross-section
values (Fd) are ꢁ15%.
10ꢀ50 cm4 photonꢀ1). This predicts a very large TP turn-on re-
sponse upon NO-induced ANO1-T formation, as observed
in the one-photon experiment (see above). These results al-
lowed us to detect NO in cells and tissues labeled with
ANO1. Moreover, the Fdmax value of ANO1-T is significant-
ly larger than those of other A-derived TP probes, despite
their similar structures.[30,31] This outcome could be ex-
plained with the photophysical properties of A and B. As
shown in Table 1, the F and Fdmax values are both larger for
B than for A by 1.8-fold, which indicates that the larger
Fdmax value of B is due to the larger F value. The cyclic
structure of the proline moiety may have reduced the vibra-
tional relaxation pathways, thereby increasing the F and
Fdmax values. This result underlines the advantage of intro-
ducing prolinamide as the donor and linker in the design of
TP turn-on probes.
Figure 2. a) Fluorescence responses of 2 mm ANO1 to various reactive
oxygen and nitrogen species (100 mm) and to AA and DHA (1 mm).
b) Effect of the pH value on the one-photon fluorescence intensity of
The TPEF spectrum from ANO1-labeled Raw 264.7
murine macrophage cells was symmetrical and slightly blue-
shifted (lfl =490 nm) relative to that measured in PBS (Fig-
*
2 mm ANO1 ( ) and ANO1-T (&) in universal buffer (0.1m citric acid,
0.1m KH2PO4, 0.1m Na2B4O7, 0.1m tris(hydroxymethyl)aminomethane
(Tris), 0.1m KCl). Excitation at l=370 nm.
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H. M. Kim, B. R. Cho et al.
Figure 4. a,c) TPM images of ANO1-labeled (3 mm) Raw 264.7 cells.
b) Two-photon excited-fluorescence spectrum measured from (a). d) The
relative TPEF intensity from the marked areas A–C in (c) as a function
of time. The digitized intensity was recorded with 1.63 s intervals for the
duration of 1h by using xyt mode. The TPEF intensities were collected at
425–575 nm upon excitation at 750 nm with fs pulses. Cells shown are
representative images from replicate experiments (n=5). Scale bar:
20 mm.
ure 4b). This indicates that the polarity of the probe envi-
ronment is rather homogeneous and slightly more hydro-
phobic than that of the PBS. Therefore, we detected the
TPEF by using a detection window of 425–575 nm. Also, the
TPEF intensity at a given spot on the ANO1-labeled cells
remained nearly the same after continuous irradiation with
the fs pulses for 60 min, which indicated the high photosta-
bility of the ANO1 (Figure 4d).
Figure 5. a) TPM images of ANO1-labeled (3 mm) Raw 264.7 cells collect-
ed at 425–525 nm. b) OPM image of LTR-labeled macrophages collected
at 575–660 nm. c) Co-localized image of (a) and (b). d and g) TPM
images of Raw 264.7 cells labeled with d) 3 mm ANO1 and g) 3 mm
ANO1-T, respectively, for 30 min. e) Cells were pretreated with
1.25 mgmLꢀ1 LPS and 200 UmLꢀ1 IFN-g for 9 h and then incubated with
ANO1 for 30 min. f) Cells were pretreated with 10 mm l-NNA,
1.25 mgmLꢀ1 LPS, and 200 UmLꢀ1 IFN-g for 9 h and then incubated with
ANO1 for 30 min. Cells shown are representative images from replicate
experiments (n=5). h) Average TPEF intensities in (d)–(g). Images of a
rat hippocampal slice stained with 20 mm ANO1: i) Bright-field image of
the CA1 and CA3 regions, as well as the dentate gyrus (DG), with 10ꢂ
magnification. j) TPM image with 10ꢂ magnification. 25 TPM images
were accumulated along the z direction at depths of approximately 100–
180 mm. k) Magnification at 40ꢂ in the DG region at a depth of approxi-
mately 100 mm before and after addition of 1 mm NMDA or 100 mm l-
NAME and 1 mm NMDA to the imaging solution. l) Time course of the
TPEF intensity in (k). (d)–(l) The TPEF data were collected at 425–
575 nm upon excitation at 750 nm with fs pulses. Scale bars: a,d) 20,
j) 300, and k) 75 mm.
Next, we tested the ability of ANO1 to detect NO in the
cells. The TPM image of Raw 264.7 cells labeled with
ANO1 was bright (Figure 4 and Figure 5). The fluorescence
intensity of ANO1 increases slightly at pH<5 in the univer-
sal buffer, so there is a possibility that the probe being locat-
ed in acidic compartments might be responsible for the
bright image. To assess such a possibility, Raw 264.7 cells
were co-labeled with ANO1 and LysoTracker Red (LTR),[36]
a well-known OP probe for acidic vesicles, and the TPM
image was colocalized with the OPM image (Figure 5a–c
and Figure S6 in the Supporting Information). The Pearsonꢀs
colocalization coefficient (A), which describes the correla-
tion of the intensity distribution between the channels,[37]
was calculated by using Autoquant X2 software. The A
value of ANO1 with LTR was 0.18 (Figure 5c). This indi-
cates that ANO1 is partially localized in the acidic compart-
ments. Moreover, when the cells were pretreated with bacte-
rial lipopolysaccharide (LPS) and interferon-g (IFN-g),
which are well-known external activators that promote NO
production by inducible nitric oxide synthase (iNOS),[9] the
TPEF intensity increased abruptly (Figure 5d,e). The aver-
age TPEF intensity lies between those measured in ANO1-
and ANO1-T-labeled cells (Figure 5 h). Moreover, the
increase
was
suppressed
by
addition
of
l-NG-nitroarginine (l-NNA), an iNOS inhibitor,[38] which
confirmed that the bright signals in the TPM images reflect
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Two-Photon Probe for Nitric Oxide in Living Tissues
FULL PAPER
enhancement in response to NO, and a maximum TP-action
cross-section of 170 GM upon reaction with excess NO. This
probe can be easily loaded into cells and can real-time mon-
itor the NO in living tissues at 100–180 mm depth for a long
period of time with minimum interference from other bio-
logically relevant species through the use of TPM.
Experimental Section
Figure 6. Viability of Raw 264.7 cells in the presence of ANO1 as meas-
ured by using a CCK-8 kit. The cells were incubated with 0, 3, 5, and
10 mm ANO1 for 6 h.
Synthesis of ANO1 and ANO1-T: Compound B was prepared by the lit-
erature method,[29] and the synthesis of the other compounds is described
below.
Synthesis of C: Compound B (0.38 g, 1.34 mmol), 2-nitro-1,4-phenylene-
diamine (0.17 g, 1.12 mmol), 1,3-dicyclohexylcarbodiimide (DCC, 0.29 g,
1.40 mmol), and 1-hydroxy-1H-benzotriazole (0.15 g, 1.12 mmol) were
dissolved in CH2Cl2 (30 mL). The reaction mixture was stirred at room
temperature for three days under a nitrogen atmosphere. The solvent
was evaporated, and the product was dissolved in CH3CN (15 mL). The
byproduct urea was removed by filtration, and the filtrate was concen-
trated under reduced pressure. The crude product was purified by
column chromatography with 5% methanol in CHCl3 as the eluent to
produce C as an orange, foam-like solid; yield 0.27 g (48%); m.p. 2708C;
1H NMR (400 MHz, CDCl3): d=8.55 (d, J=1.6 Hz, 1H), 8.23 (s, 1H,
amide NH), 8.08 (d, J=2.8 Hz, 1H), 7.83 (dd, J=8.8, 1.6 Hz, 1H), 7.76
(d, J=8.8 Hz, 1H), 7.57 (dd, J=9.2, 2.8 Hz, 1H), 7.56 (d, J=8.8 Hz, 1H),
7.00 (dd, J=8.8, 2.0 Hz, 1H), 6.84 (d, J=2.0 Hz, 1H), 6.70 (d, J=9.2 Hz,
1H), 6.12 (s, 2H, amine NH2), 4.27 (t, J=5.8 Hz, 1H), 3.87 (m, 1H), 3.39
(m, 1H), 2.61 (s, 3H), 2.38 (m, 2H), 2.14 ppm (m, 2H); 13C NMR
(100 MHz, [D6]-dimethylsulfoxide ([D6]DMSO)): d=195.6, 169.9, 144.9,
140.1, 135.0, 129.2, 128.7, 128.2, 127.7, 124.7, 124.2, 123.8, 122.5, 117.0,
115.1, 114.3, 111.1, 104.8, 62.58, 47.87, 29.74, 24.53, 22.24 ppm.
the presence of NO (Figure 5 f,h). Furthermore, ANO1 was
found to be nontoxic to cells during the imaging experi-
ments, as determined by using a Cell Counting Kit 8 (CCK-
8) assay (Figure 6). These results establish that ANO1 is ca-
pable of detecting NO in living cells with minimum interfer-
ence from pH value and cytotoxicity.
We further investigated the utility of ANO1 in live-tissue
imaging. TPM images were obtained from a slice of 14-day-
old rat hippocampal tissue incubated with 20 mm ANO1 for
1 h at 378C. It takes a longer time to stain the living tissues,
during which time they may be deformed, so an excess
amount (20 mm) of ANO1 was used to facilitate staining.
The slice from the brain was too large to show with one
image, so two images were obtained in each xy plane and
combined. The bright-field image revealed the CA1 and
CA3 regions and also the dentate gyrus (DG; Figure 5i). As
the structure of the brain tissue is known to be inhomogene-
ous in its entire depth, we accumulated ten TPM images at
depths of 100–180 mm to visualize the distribution of NO.
They reveal intense fluorescence in the DG and CA3 re-
gions (Figure 5j).[39] The image obtained at a higher magnifi-
cation clearly shows the distribution of NO in the DG
region (Figure 5k). When 1 mm N-methyl-d-aspartic acid
(NMDA), a reagent that promotes NO production by con-
stitutive nitric oxide synthase (cNOS),[39,40] was added to the
imaging solution, the TPEF intensities increased immediate-
ly. Moreover, the increase was suppressed by the addition of
N-(G)-nitro-l-arginine methyl ester (l-NAME), a cNOS in-
hibitor,[39,40] which confirmed that the bright signals in the
TPM images reflect the presence of NO (Figure 5k,l). Fur-
thermore, the TPM images obtained at a depth of 100–
180 mm revealed the NO distribution in the given plane
along the z direction in the CA1 and DG regions (Figure S7
in the Supporting Information). These findings demonstrate
that ANO1 is capable of detecting NO at a depth of 100–
180 mm in living tissues by using TPM.
Synthesis of ANO1: Hydrochloric acid (1 mL) was added dropwise to a
solution of C (0.20 g, 0.48 mmol) in CH2Cl2 (10 mL) at 08C. The reaction
mixture was stirred for 30 min. SnCl2·2H2O (1.4 g, 6.21 mmol) was added
to this mixture, and stirring was continued overnight at room tempera-
ture. The resulting mixture was washed with 2n KOH and extracted with
CH2Cl2. The organic phase was dried over Na2SO4, filtered, and evapo-
rated. The crude product was purified by column chromatography with
5% methanol in CHCl3 as the eluent to afford a yellow powder; yield
0.14 g (75%); m.p. 1808C; 1H NMR (400 MHz, CDCl3): d=8.32 (d, J=
1.6 Hz, 1H), 7.96 (s, 1H, amide NH), 7.93 (dd, J=8.8, 1.6 Hz, 1H), 7.82
(d, J=9.2 Hz, 1H), 7.65 (d, J=8.8 Hz, 1H), 7.06 (dd, J=9.2, 2.4 Hz, 1H),
7.05 (s, 1H), 6.93 (d, J=2.4 Hz, 1H), 6.55 (s, 2H), 4.25 (t, J=5.8 Hz,
1H), 3.86 (m, 1H), 3.43 (m, 1H), 3.35 (brs, 4H, diamine 2NH2), 2.66 (s,
3H), 2.40 (m, 2H), 2.13 ppm (m, 2H); 13C NMR (100 MHz, CDCl3): d=
197.6, 171.1, 147.2, 137.3, 135.6, 131.7, 131.3, 130.4, 126.6, 126.2, 124.9,
117.2, 116.7, 112.1, 109.3, 107.2, 65.1, 50.2, 31.9, 30.0, 26.8 ppm; HRMS
(FAB+): m/z calcd for [C23H25N4O2]: 389.1978; found: 389.1978.
Synthesis of ANO1-T: Sodium nitrite (NaNO2, 0.25 g, 3.6 mmol) was
added dropwise to a mixture of ANO1 (0.14 g, 0.36 mmol) in HCl (aq,
pH 2) solution over a period of 30 min at 08C. The mixture was stirred
for 1 h at room temperature. The crude product was extracted with
CH2Cl2, washed with water in twice, and evaporated. The residue was pu-
rified by column chromatography with ethyl acetate/hexane (4:1) as the
eluent to afford a dark yellow, foam-like solid; yield: 0.072 g (51%); m.p.
1
1558C; H NMR (400 MHz, CDCl3): d=8.49 (d, 1H, amide NH), 8.34 (d,
J=1.6 Hz, 1H), 7.95 (dd, J=8.8, 1.6 Hz, 1H), 7.86 (d, J=8.8 Hz, 1H),
7.83 (brd, J=9.2 Hz, 1H), 7.68 (d, J=8.8 Hz, 1H), 7.10 (dd, J=8.8,
2.4 Hz, 1H), 7.00 (brs, 1H), 6.98 (d, J=2.4 Hz, 1H), 4.36 (t, J=6.0 Hz,
1H), 3.96 (m, 1H), 3.48 (m, 1H), 2.66 (s, 3H), 2.48 (m, 2H), 2.16 ppm
(m, 2H); 13C NMR (100 MHz, [D6]DMSO): d=196.6, 172.0, 146.6, 136.9,
130.7, 130.4, 130.0, 125.6, 124.5, 124.0, 116.3, 104.7, 62.18, 48.69, 31.45,
26.42, 23.52 ppm; HRMS (FAB+): m/z calcd for [C23H22N5O2]: 400.1774;
found: 400.1773.
Conclusion
We have developed a small-molecule TP turn-on probe
(ANO1) that shows a rapid and specific NO response, pH
insensitivity in the biologically relevant range, strong TPEF
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H. M. Kim, B. R. Cho et al.
Spectroscopic measurements: Absorption spectra were recorded on a S-
3100 UV/Vis spectrophotometer, and fluorescence spectra were obtained
with a FluoroMate FS-2 fluorescence spectrophotometer with a 1 cm
standard quartz cell. The fluorescence quantum yield was determined by
using coumarin 307 (F=0.95 in MeOH) as the reference by the literature
method.[41] Reactive oxygen and nitrogen species (100 mm unless other-
wise stated) were administered to ANO1 in PBS (pH 7.4) as follows.
Nitric oxide (NO) was used from a stock solution (1.9 mm), prepared by
purging PBS (0.01m, pH 7.4) with N2 gas for 30 min and then with NO
(99.5%) for 30 min.[42] HNO was delivered from Na2N2O3 (Angeliꢀs salt,
Cayman Chemical). It has been reported that Angeliꢀs salt is a precursor
of HNO and NO, so Angeliꢀs salt stock solution was made in deoxygenat-
ed PBS, which was prepared by purging with argon gas for 30 min, to pre-
vent NO production.[43] Superoxide (O2ꢀ), nitrite (NO2ꢀ), and nitrate
(NO3ꢀ) were delivered from KO2, NaNO2, and NaNO3 (Aldrich), respec-
tively. H2O2, tert-butylhydroperoxide (TBHP), and hypochlorite (NaOCl)
were delivered from 30, 70, and 10% aqueous solutions, respectively. Hy-
10 mW average power in the focal plane. To obtain images in the 425–
575 nm range, internal photomultiplier tubes were used to collect the sig-
nals in an 8-bit unsigned 512ꢂ512 and 1024ꢂ1024 pixels at 800 and
400 Hz scan speeds, respectively.
Photostability: The photostability of ANO1 was determined by monitor-
ing the changes in TPEF intensity with time at three designated positions
of ANO1-labeled (3 mm) Raw 264.7 cells chosen without bias. The TPEF
intensity remained nearly the same for one hour (Figure 4d), which indi-
cated high photostability.
Detection window: The TPM image of Raw 264.7 cells labeled with 3 mm
ANO1 was bright. Moreover, the TP excited-fluorescence (TPEF) spec-
trum from the white-circled domain in Figure 4a was symmetrical and
slightly blueshifted from that measured in PBS, with an emission maxi-
mum at approximately 490 nm (Figure 4b). This indicates that the polari-
ty of the probe environment is rather homogeneous and slightly more hy-
drophobic than that of PBS. Therefore, NO was detected by using a de-
tection window of 425–575 nm. For colocalization experiments, the emis-
sion spectra of ANO1 and LysoTracker Red in Raw 264.7 cells were com-
pared. The detection windows for ANO1 and LysoTracker Red were
determined by considering two factors: 1) the emission from the two
probes should be separated as far as possible and 2) the emission intensi-
ties from the probes should be similar. The detection windows that meet
the above requirements were 425–525 (ANO1) and 575–660 nm (Lyso-
Tracker Red), respectively (Figure S6 in the Supporting Information).
C
C
droxyl radicals ( OH) and tert-butoxy radicals ( OtBu) were generated by
the reaction of 1 mm Fe2+ with 200 mm H2O2 or TBHP, respectively.
Sodium peroxynitrite (Cayman Chemical) was prepared in aqueous stock
solution in Millipore water.[44]
Solubility of ANO1 in PBS: A small amount of dye was dissolved in
DMSO to prepare the stock solutions (1.0ꢂ10ꢀ2 m). The solution was di-
luted to (6.0ꢂ10ꢀ3–6.0ꢂ10ꢀ5)m and added to a cuvette containing PBS
(3.0 mL, pH 7.4) by using a microsyringe. In all cases, the concentration
of DMSO in buffer was maintained as 0.2%.[28] The plots of the fluores-
cence intensity against the total amount of dye injected into the cuvette
were linear with low dye content and showed downward curvature as
more dye was added (Figure S2 in the Supporting Information). The
maximum point in the linear region was taken as the solubility. The solu-
bility of ANO1 and ANO1-T in PBS was 4.0 mm.
Cell viability: To confirm that the probe couldn’t affect the viability of
Raw 264.7 cells under the experimental incubation conditions, a CCK-8
kit (Dojindo, Japan) was used according to the manufacturerꢀs protocol.
The results are shown in Figure 6.
Effects of LPS, IFN-g, NMDA, and l-NAME: The stock solution of
10 mm NMDA (Sigma–Aldrich) was prepared by dissolving NMDA
(1.5 mg) in distilled H2O (1.0 mL), and the solution of 100 mm l-NAME
(Sigma–Aldrich) was prepared by dissolving l-NAME·HCl (27 mg) in
H2O (1.0 mL). Each solution (1 mL) was diluted to 0.1 mL with artificial
cerebrospinal fluid (ACSF; 138.6 mm NaCl, 3.5 mm KCl, 21 mm NaHCO3,
0.6 mm NaH2PO4, 9.9 mm d-glucose, 1 mm CaCl2, and 3 mm MgCl2) and
added to the imaging solution (0.9 mL) with a micropipette. The stock
solution of LPS (Sigma–Aldrich) was prepared by dissolving LPS (1 mg)
in DPBS (1.0 mL). The solution (1.25 mL) was diluted into imaging cell
medium (1.0 mL). The stock solution of IFN-g (Sigma–Aldrich) was pre-
pared by dissolving 100 mg of IFN-g in DPBS (1 mL). The solution was
diluted by 10% in DPBS. The final stock solution of IFN-g (2 mL) was
added to the imaging cell medium (1.0 mL).
Measurement of two-photon cross-section: The two-photon cross-section
(d) was determined by using the femtosecond fluorescence measurement
technique previously described.[45] ANO1 and ANO1-T (1.0ꢂ10ꢀ6 m)
were dissolved in 30 mm PBS (pH 7.4), and the two-photon induced fluo-
rescence intensity was measured at 720–880 nm by using rhodamine 6G
as the reference, because the two-photon properties of this compound
have been well characterized in the literature.[46] The intensities of the
two-photon induced fluorescence spectra of the reference and sample
emitted at the same excitation wavelength were determined. The TPA
cross-section was calculated by using d=dr(SsFrFrcr)/(SrFsFscs), in which
the subscripts s and r stand for the sample and reference molecules, the
intensity of the signal collected by a CCD detector was denoted as S, F
is the fluorescence quantum yield, f is the overall fluorescence collection
efficiency of the experimental apparatus, the number density of the mole-
cules in solution was denoted as c, and dr is the TPA cross-section of the
reference molecule.
Preparation and staining of fresh rat hippocampal slices: Slices were pre-
pared from the hippocampi of 2-week-old rats (SD). Coronal slices were
cut to 400 mm thick by using a vibrating-blade microtome in ACSF
(138.6 mm NaCl, 3.5 mm KCl, 21 mm NaHCO3, 0.6 mm NaH2PO4, 9.9 mm
d-glucose, 1 mm CaCl2, and 3 mm MgCl2). Slices were incubated with
20 mm ANO1 in ACSF bubbled with CO2/air (5:95) for 1 h at 378C. Slices
were then washed three times with ACSF, transferred to 0.17 mm delta T
dishes (Bioptechs Inc.), and observed in a spectral confocal multiphoton
microscope. The TPM images of fresh rat hippocampal slices labeled
with 20 mm ANO1 obtained at 100–180 mm depth are shown in Figure S7
in the Supporting Information.
Cell culture and imaging: Raw 264.7 cells (American Type Culture Col-
lection (ATCC), Manasas, VA, USA) were cultured in Dulbeccoꢀs modi-
fied Eagleꢀs medium (DMEM, WelGene Inc., Seoul, Korea) supplement-
ed with 10% fetal bovine serum (FBS, WelGene) and penicillin
(100 unitsmLꢀ1). Two days before imaging, the cells were passaged and
plated on 0.17 mm delta T dishes (Bioptechs Inc.). The cells were incu-
bated in a humidified atmosphere of CO2/air (5:95) at 378C. For labeling,
the growth medium was removed and replaced with Dulbeccoꢀs phos-
phate buffer (DPBS, WelGene) containing calcium and magnesium. The
cells were treated with 1 mm ANO1 or ANO1-T (3 mL) in DMSO stock
solution (3 mm ANO1, ANO1-T) and then incubated for 30 min. After
staining, the cells were washed three times with DPBS and then imaged.
Acknowledgements
This work was supported by the Korea Healthcare Technology R&D
Project, Ministry of Health & Welfare, Republic of Korea (A111182),
NRF grants (nos.: 2011-0028663, 2011-0004321, and 2011-0018396), and
an Ajou University research fellowship of 2011.
Two-photon fluorescence microscopy: Two-photon fluorescence micro-
scopy images of ANO1- and ANO1-T-labeled cells and tissues were ob-
tained with spectral confocal and multiphoton microscopes (Leica TCS
SP2) with ꢂ10, ꢂ40 dry, and ꢂ100 oil objectives (numerical aperture:
NA =0.30, 0.75, and 1.30, respectively). The two-photon fluorescence mi-
croscopy images were obtained with a DM IRE2 Microscope (Leica) by
exciting the probes with a mode-locked titanium-sapphire laser source
(Coherent Chameleon, 90 MHz, 200 fs) set at a wavelength of 750 nm
and output power of 1345 mW, which corresponded to approximately
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Revised: June 21, 2012
Published online: && &&, 0000
Chem. Eur. J. 2012, 00, 0 – 0
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H. M. Kim, B. R. Cho et al.
Fluorescent Probes
E. W. Seo, J. H. Han, C. H. Heo,
J. H. Shin, H. M. Kim,*
B. R. Cho* ..................... &&&& — &&&&
A Small-Molecule Two-Photon Probe
for Nitric Oxide in Living Tissues
NO lightens up: A small-molecule
two-photon (TP) probe (ANO1; see
scheme) is reported that shows a rapid
and specific nitric oxide (NO)
response, in the form of a strong TP
excited-fluorescence enhancement in
response to NO, with a maximum TP-
action cross-section of 170 GM (GM:
10ꢀ50 cm4 photonꢀ1). The probe can be
easily loaded into cells and can real-
time monitor NO in living tissues.
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These are not the final page numbers!