Seminaphthofluorescein-based fluorescent probes for imaging nitric oxide in live cells

Article abstract of DOI:10.1021/ic200986v

Fluorescent turn-on probes for nitric oxide based on seminaphthofluorescein scaffolds were prepared and spectroscopically characterized. The Cu(II) complexes of these fluorescent probes react with NO under anaerobic conditions to yield a 20-45-fold increase in integrated emission. The seminaphthofluorescein-based probes emit at longer wavelengths than the parent FL1 and FL2 fluorescein-based generations of NO probes, maintaining emission maxima between 550 and 625 nm. The emission profiles depend on the excitation wavelength; maximum fluorescence turn-on is achieved at excitations between 535 and 575 nm. The probes are highly selective for NO over other biologically relevant reactive nitrogen and oxygen species including NO₃ ^-, NO₂^-, HNO, ONOO^-, NO₂, OCl^-, and H₂O₂. The seminaphthofluorescein- based probes can be used to visualize endogenously produced NO in live cells, as demonstrated using Raw 264.7 macrophages.

Full text of DOI:10.1021/ic200986v

ARTICLE
pubs.acs.org/IC
Seminaphthofluorescein-Based Fluorescent Probes for Imaging
Nitric Oxide in Live Cells
Michael D. Pluth, Maria R. Chan, Lindsey E. McQuade, and Stephen J. Lippard*
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States
S Supporting Information
b
ABSTRACT: Fluorescent turn-on probes for nitric oxide based
on seminaphthofluorescein scaffolds were prepared and spec-
troscopically characterized. The Cu(II) complexes of these
fluorescent probes react with NO under anaerobic conditions
to yield a 20ꢀ45-fold increase in integrated emission. The
seminaphthofluorescein-based probes emit at longer wave-
lengths than the parent FL1 and FL2 fluorescein-based genera-
tions of NO probes, maintaining emission maxima between 550
and 625 nm. The emission profiles depend on the excitation
wavelength; maximum fluorescence turn-on is achieved at excitations between 535 and 575 nm. The probes are highly selective for
NO over other biologically relevant reactive nitrogen and oxygen species including NO3ꢀ, NO2ꢀ, HNO, ONOOꢀ, NO2, OClꢀ, and
H2O2. The seminaphthofluorescein-based probes can be used to visualize endogenously produced NO in live cells, as demonstrated
using Raw 264.7 macrophages.
’ INTRODUCTION
NO probes is to employ transition metals to modulate the
fluorescence of a pendant fluorophore. In contrast to purely organic
NO probes, metal-based probes detect NO directly. The close
proximity of a transition metal can either quench the fluores-
cence of a pendant fluorophore until it reacts with NO or
facilitate reaction of NO with a dye component. Metal-based
fluorescent probes include those based on Co(II), Fe(II), Ru(II),
Cu(II), or Rh(II).32,36ꢀ41
Previously, we reported FL1 and FL2 fluorescent probe
families for imaging endogenous nitric oxide in live cells and tissue;
these sensors employ Cu(II) to modulate NO reactivity.25,36,42ꢀ45
The probes bind Cu(II) at an 8-aminoquinaldine unit appended to
the fluorophore. Upon treatment with NO under anaerobic
conditions, the Cu(II) ion is reduced to Cu(I) with concomitant
nitrosation of the secondary nitrogen to form the fluorescent
nitrosamine product (Figure 1).43,46 Probes from the CuFL1 and
CuFL2 families can detect endogenous NO in cell culture and live
tissue slices, emitting in the region of the spectrum typical for
fluorescein-based dyes, with maxima near 525 nm. To improve the
utility of these NO probes, scaffolds that emit at longer wavelengths
are desirable. Such probes would not only expand the palette of
colors available for multidye imaging experiments but also require
lower energy excitation and thus diminish tissue damage. Further-
more, longer wavelength probes facilitate deeper imaging in live
tissue slices or live animals. To address these goals, the FL1 scaffold
was modified to emit at longer wavelengths by extending the
fluorescein system to a seminaphthofluorescein moiety. Here, we
report the synthesis, photophysical characterization, and cellular
The multifaceted roles of biological nitric oxide (NO) have
continued to emerge since the initial discovery that NO is the
endothelium-derived relaxing factor (EDRF).1ꢀ3 Subsequent
studiesrevealedthatnitric oxide is an importantsignalingmolecule
in the immune, cardiovascular, and nervous systems.4ꢀ6 The
delicate balance of NO production, action, and translocation
requires tight regulation because a breakdown in NO homeostasis
is associated with a variety of pathological conditions ranging from
carcinogenesis to neurodegradation.7ꢀ13 In order to advance our
understanding of pivotal roles played by NO in plants, bacteria,
invertebrates, and mammals, chemists have devised strategies to
detect this important biological messenger.6,14ꢀ19 Although several
strategies are currently employed to detect NO either directly or
indirectly, the use of fluorescent probes offers distinct advantages,
including high sensitivity and convenience when applied using
common microscopic techniques, to yield valuable spatiotemporal
information about biologically generated NO.
Fluorescent probes based on small molecule fluorophores,
single-walled carbon nanotubes (SWNTs), and proteins have
been reported.20ꢀ31 Small molecule fluorescent probes typically
rely on one of two strategies for NO detection. The first strategy
utilizes organic moieties with electron-rich components, such as
1,2-diaminobenzenes, that react with an oxidation product of NO,
such as N2O3, to form an electron-deficient product, such as a
triazole, to modulate the emission of the fluorophore.22,32ꢀ35
Although such probes comprise the majority of small molecule
fluorescent sensors for NO, their inability to react with NO directly,
relying on an indirect response that requires a sufficiently
oxygenated environment to function, is a liability. The second
main strategy used in the preparation of small-molecule-based
Received: May 10, 2011
Published: September 07, 2011
r
2011 American Chemical Society
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Scheme 1. Preparation of the Seminaphthofluorescein Fa-
mily of Probes for Nitric Oxide (see Experimental Section for
conditions)
Figure 1. Schematic showing the mechanism of fluorescence response
for Cu(II)-based NO probes such as FL1 and FL2.
all probes, the emission profile changes dramatically with the
excitation wavelength (Figure 2, Supporting Information Figure
S4). For example, excitation of SNFL1 and SNFL1E between
500 and 535 nm results in an emission maximum near 545 nm
and a strong shoulder at 585 nm. Excitation at longer wavelengths
between 535 and 550 nm, however, results in weak emission at
620 nm. The emission properties of SNFL1Br and SNFL1EBr are
more complex than those of the nonbrominated analogs. At
shorter wavelength excitations, up to 525 nm, the emission
maximum is at 550 nm with a weaker band near 600 nm. Above
530 nm excitation, the principal emission band at 550 nm de-
creases and the main emission shifts to 615 nm (Figure 2).
Detailed 2D excitation/emission plots for all of the probes both
before and after reaction with NO are supplied in the Supporting
Information (Figure S4).
By integrating the emission in both the on and off states of
the probes as a function of excitation wavelength, the excitation-
dependent magnitude of fluorescence turn-on was determined.
For SNFL1, the fluorescence response to NO is maximal in the
range between 525 and 535 nm with a net turn-on of 20-fold (42-
fold for SNFL1E). SNFL1Br reaches a maximum turn-on of 22-
fold (36-fold for SNFL1EBr) after reaction with NO near
535ꢀ575 nm. For comparison with the magnitude of turn-on using
previously reported NO probes, a similar analysis was performed
for FL2. A fluorescence turn-on of 23-fold had been observed
using an excitation wavelength of 470 nm. Measurements of the
fluorescence turn-on as a function of excitation revealed that, at
λex = 515 nm, there is a 92-fold increase in integrated emission.
Selectivity for NO over other RONS. Because a variety of
reactive oxygen and nitrogen species (RONS) involved in biolo-
gical processes are associated with nitric oxide, the NO selectivity
of the seminaphthofluorescein probes was determined (Figure 3).
Because of the excitation wavelength dependence of the turn-on
with NO, the response to RONS was monitored at various
excitation wavelengths (Figure 3, top). For comparison between
the probes, λex = 530 nm was used (Figure 3, bottom). Addition
of either NO or S-nitroso-N-acetyl-DL-penicillamine (SNAP), a
common NO donor, resulted in a large 20ꢀ45-fold increase in
integrated emission. This turn-on with 100 equiv of SNAP was
identical to that observed after treatment with 1300 equiv of NO.
imaging experiments of a family of seminaphthofluorescein-based
SNFL probes that are selective for nitric oxide.
’ RESULTS AND DISCUSSION
To extend the emission wavelengths of the FL1 family of NO
probes, we adopted similar synthetic methodologies to extend the
π-system by incorporation of a seminaphthofluorescein moiety.
Seminaphthofluorescein dyeshave been usedas fluorescent probes
for numerous biological applications including metal ion detection
and pH measurements.47ꢀ53 Such dyes typically emit between
550 and 625 nm, values compatible with common yellow or
orange filter sets in fluorescence microscopes.
Synthesis. The first step in the synthesis of the seminaphtho-
fluorescein NO probes involved bromination of the benzoate de-
rivative 1 to form the desired bromomethyl species 3 (Scheme 1).53
During this reaction, we noticed that an excess of brominating agent
2 and extended times resulted in overbromination to yield 4. The
position of the bromine atom was confirmed by 2D NMR spec-
troscopy and single crystal X-ray crystallography (Supporting
Information Figures S1ꢀS3, Table S1). Kornblum oxidation of
the bromomethyl derivative 3 and 4 yielded the respective alde-
hydes 5 and 6, which were coupled to substituted 8-aminoquinal-
dines (7, 8) by reductive amination to yield the final constructs
SNFL1, SNFL1E, SNFL1Br, and SNFL1EBr.
Photophysical Properties. The seminaphthofluorescein
probes show two overlapping bands in the UVꢀvis spectrum at
∼495/520 nm for SNFL1 (ε496 = 8500 ( 100 Mꢀ1 cmꢀ1
,
ε517 = 8700 ( 100 Mꢀ1 cmꢀ1) and SNFL1E (ε496 = 5030 ( 40
M
ꢀ1 cmꢀ1, ε519 = 5200 ( 50 Mꢀ1 cmꢀ1) and at ∼505/530 nm for
the brominated analogs SNFL1Br (ε505 = 4450 ( 50 Mꢀ1 cmꢀ1
,
ε530 = 4650 ( 40 Mꢀ1 cmꢀ1) and SNFL1EBr (ε501 = 4070 ( 40
M
ꢀ1 cmꢀ1, ε532 = 3930 ( 30 Mꢀ1 cmꢀ1). The Cu(II) complexes
display slight bathochromic shifts in absorbance maxima but other-
wise have photophysical properties similar to those of the free ligands
(Table 1). Excitation at either of the absorbance maxima results in
weak emission in the range 535ꢀ545 nm, corresponding to the off-
states of the probes.
Upon reaction with nitric oxide, the fluorescence of the Cu(II)-
bound seminaphthofluorescein probes increased significantly. For
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Table 1. Photophysical Properties of Seminaphthofluorescein NO Probes and Representative Properties of FL1 and FL2 Probes
for Comparisona
absorption λmax (nm), ε (ꢁ10ꢀ4 Mꢀ1 cmꢀ1
)
emission λmax (nm), Φb(%)
unbound
+Cu(II)c
unbound
+Cu(II)c
+NOd
FL136
FL1E45
FL225
504, 4.2 ( 0.1
499, 4.0 ( 0.1
520, 7.7 ( 0.2
520, 2.57 ( 0.02
515, 0.74 ( 0.05
522, 0.37 ( 0.05
542, 2.7 ( 0.4
544, 2.1 ( 0.1
541, 0.9 ( 0.1
548, 0.72 ( 0.07
544, 1.2 ( 0.2
546, 0.9 ( 0.1
0.49 ( 0.06e
520, not reported
520, 3.37 ( 0.08
512, 0.76 ( 0.04
522, 0.72 ( 0.04
538, 2.1 ( 0.1
526, 58 ( 2
526, 22 ( 1
526, 51 ( 7
526, 40 ( 8
548h 22 ( 1
506, 2.08 ( 0.05
498, 2.91 ( 0.07
500, 1.79 ( 0.07
496, 0.85 ( 0.01
517, 0.87 ( 0.01
496, 0.503 ( 0.004
519, 0.520 ( 0.005
505, 0.445 ( 0.005
530, 0.465 ( 0.004
501, 0.407 ( 0.004
532, 0.393 ( 0.003
500, 0.77 ( 0.01
494, 1.14 ( 0.07
496, 1.12 ( 0.06
502, 0.468 ( 0.006
527, 0.507 ( 0.006
504, 0.203 ( 0.002
522, 0.408 ( 0.004
504, 0.477 ( 0.007
540, 0.54 ( 0.01
505, 0.47 ( 0.01
536, 0.51 ( 0.01
FL2E44
SFNL1
543, 0.96 ( 0.04
538, 0.92 ( 0.09
543, 0.59 ( 0.04
542, 0.34 ( 0.02
546, 0.14 ( 0.01
0.34 ( 0.01f
SNFL1E
SNFL1Br
SNFL1EBr
549h 24 ( 2
548/597g,i,j 14 ( 1
549/597g,i,j 13 ( 1
0.25 ( 0.03e
0.12 ( 0.01f
a Spectroscopic measurements were performed in 50 mM PIPES and 100 mM KCl buffer at pH 7.0 at 25 °C. b Quantum yields are based on fluorescein
(Φ = 0.95 in 0.1 N NaOH) or rhodamine B (Φ = 0.54 in H2O). c One equivalent of CuCl2 was added per copper-binding site. d 1300 equiv of NO, 60 min at
37 °C. Complexes generated in situ. e Broad emission, λmax ( 5 nm. f The broad emission prevented meaningful λmax determination. g λmax(UV) = λex
=
517 nm. h λmax(UV) = λex = 527 nm. i The two emission maxima are of approximately equal intensity. j λex g 530 nm results in an emission maxima at 615 nm.
Furthermore, all probes reached a maximum and constant
1, 5, 10, 50, 100, 500, or 1000 equiv of Zn(II) to a 10 μM solution
of the Cu(II)-bound seminaphthofluorescein probes resulted in
only negligible fluorescence. Even at 10 mM Zn(II), only a 5.5-
and 2.5-fold turn-on, for CuSNFL1 and CuSNFL1Br, respec-
tively, was observed. These results reveal that the seminaphtho-
fluorescein probes are more sensitive to NO than to biological
concentrations of Zn(II).
fluorescent state within 1 h of treatment with either SNAP or
ꢀ
NO. By contrast, treatment with RONS such as NO3ꢀ, NO2
,
ONOOꢀ, HNO, H2O2, or OClꢀ did not result in fluorescence
enhancement. Nitrogen dioxide (NO2), an oxidation product of
NO that can directly nitrosate secondary amines, afforded minimal
fluorescence turn-on. These results demonstrate the high selec-
tivity of the seminaphthofluorescein probes for NO over other
potential RONS in biological systems.
Fluorescence Imaging of Endogenous NO. Having demon-
strated the efficacy of the seminaphthofluorescein-based probes
for NO detection in the cuvette, we next evaluated their biological
compatibility by imaging NO produced in live cells. For these
studies we investigated Raw 264.7 murine macrophage cells,
which generate NO from iNOS upon stimulation with endotox-
ins and cytokines.58 Upon addition of lipopolysaccharide (LPS)
and interferon-γ (INF-γ), Raw 264.7 cells produce micromolar
concentrations of NO and are therefore ideal for evaluating the
potential of the seminaphthofluorescein-based probes under bio-
logical conditions. Treatment of the cells with only the Cu(II)-
bound fluorescent probes resulted in minimal observed emission.
By contrast, stimulation with LPS and INF-γ in the presence of
the seminaphthofluorescein probes resulted in a significant in-
crease in fluorescence intensity. In all cases, a substantial emission
enhancement occurred, thus demonstrating the suitability of the
probes for detection of endogenous levels of NO. Representative
images of the fluorescence both with and without stimulation of
iNOS are presented in Figure 6. Sample line scans showing the
magnitude of signal in both sets of cells are also included. Despite
the ester functionality on the SNFL1E and SNFL1EBr probes,
which isoften used to impartcell trappability effected by hydrolysis
by intracellular esterases to yield negatively charged carboxylates,
the esterified seminaphthofluorescein probes diffused out of the
cells in a manner similar to that of the parent SNFL1 and SNFL1Br
probes (see Supporting Information Figures S11ꢀS14).
pH Dependence. Much like the fluorescein-based FL1 and
FL2 scaffolds, the emission of the seminaphthofluorescein scaf-
folds is pH-dependent. To examine the nature of this depen-
dence, pH titrations were performed for SNFL1 and SNFL1Br
using both UVꢀvis and fluorescence spectroscopy. The pH
titrations were performed for the metal-free forms of the ligands
to remove the Cu-binding equilibrium from the measurement.
For both probes, the emission was highest in the 6ꢀ8 pH range, a
region compatible for most biological experiments (Figure 4,
left). For SNFL1, the UVꢀvis spectrum revealed a pKa of 7.3
(Figure 4, right). Similarly, measuring the integrated fluores-
cence as a function of pH revealed pKa values of 4.9, 6.3, and 7.5
(Supporting Information Figure S9). For the SNFL1Br probe, the
UVꢀvis pH titration yielded a pKa of 7.5, and values of 5.5, 6.8, and
7.5 were determined by fluorescence spectroscopy (Supporting
Information Figure S10). Although the fluorescence of
SNFL1 and SNFL1Br changes in the physiological pH range
6ꢀ8, the magnitude of the change in emission, ∼2-fold, is an
order of magnitude less than the fluorescence response of the
probe to NO.
Influence of Zn(II) on Fluorescence. Because of the similarity
of the seminaphthofluorescein scaffolds to previous Zn(II) sensors
developed in our laboratory53,54 and because NO mediates Zn(II)
release from metallothioneins,55ꢀ57 we investigated the potential
of Zn(II) to elicit a fluorescence response from the seminaphtho-
fluorescein-based NO probes. In particular, we titrated CuSNFL1
and CuSNFL1Br with Zn(II) and monitored the integrated
emission as a function of added Zn(II) (Figure 5). Addition of
Summary. The synthesis and photophysical properties of
seminaphthofluorescein-based fluorescent probes for nitric oxide
are reported. Upon reaction with NO, the Cu(II) ligated SNFL1,
SNFL1E, SNFL1Br, and SNFL1EBr probes exhibit 20ꢀ45-fold
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Figure 2. Emission of (a) CuSNFL1 and (b) CuSNFL1Br before and
after addition of NO as a function of excitation wavelength. The excitation
peaks have been removed for clarity. (c) Integrated fluorescence turn-on
as a function of excitation wavelength for CuSNFL1 and CuSNFL1Br. (d)
Integrated emission of Cu2FL2 as a function of wavelength is shown for
comparison. Conditions: 1 μM probe, 50 mM PIPES, 100 mM KCl, pH 7,
37 °C, 1300 equiv of NO incubated for 1 h.
Figure 3. (Top) Selectivity of CuSNFL1 fluorescence response for NO
over other reactive nitrogen and oxygen species as a function of excitation
wavelength. (Bottom) Comparison of the selectivity for NO over other
RONS for CuSNFL1, CuSNFL1Br, CuSNFL1E, and CuSNFL1EBr.
Conditions: 50 mM PIPES, 100 mM KCl, pH 7.0, 37 °C, 60 min, 100
equiv of RONS or SNAP, 1300 equiv of NO, λex = 530 nm. The selectivity
of the probes over a broad range of excitation wavelengths is reported in
the Supporting Information (Figures S5ꢀS8 and Tables S2ꢀS5).
increases in integrated fluorescence. All of the probes are highly
selective for NO or NO donors over other reactive oxygen and
nitrogen species. Treatment of the Cu(II)-bound ligands with a
large excess of Zn(II) did not elicit a significant fluorescence
response. All of the seminaphthofluorescein probes are cell perme-
able and can be used to image endogenous levels of NO, as
demonstrated in Raw 264.7 murine macrophage cells.
’ EXPERIMENTAL SECTION
Synthetic Materials and Methods. Seminaphthofluorescein
precursors 1, 3, and 5,53 aminoquinaldines 759 and 8,60 and FL225 and
FL2E44 were prepared as described previously. Silica gel (SiliaFlash F60,
Silicycle, 230ꢀ400 mesh) was used for column chromatography. Thin-
layer chromatography (TLC) was performed on J. T. Baker 1BꢀF silica
gel plates (250 μm thickness) and viewed by UV illumination. Preparative
TLC was performed on Silicycle SiliaPlates (1000 μm thickness). NMR
spectra were acquired on a Bruker 400 MHz spectrometer at ambient
temperature. Chemical shifts are reported in parts per million (δ) and
are referenced to residual protic solvent resonances. The following
abbreviations are used in describing NMR couplings: (s) singlet, (d)
doublet, (t) triplet, (q) quartet, (b) broad, (m) multiplet. Melting
points were measured on a MelTemp apparatus and are reported as
Figure 4. (Left) Integrated fluorescence as a function of pH for SNFL1
and SNFL1Br. (Right) Change in absorbance in the UVꢀvis spectrum at
505 nm for SNFL1 and SNFL1Br as a function of pH. Conditions: 20 μM
dye, 100 mM KCl, 25 °C.
uncorrected temperatures. High-resolution mass spectra were measured
by the staff at the MIT Department of Chemistry Instrumentation
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Facility (DCIF). Elemental analyses were performed by a commercial
analytical laboratory.
Master 4 L-format scanning spectrofluorimeter (Photon Technology
International) at 25.0 ( 0.1 or 37.0 ( 0.1 °C. All fluorescence measure-
ments were made under anaerobic conditions, with cuvette solutions
prepared in an inert atmosphere glovebox. All UVꢀvis and fluorescence
experiments were repeated at least in triplicate.
Spectroscopic Materials and Methods. Piperazine-N,N0-bis(2-
ethanesulfonic acid) (PIPES, Calbiochem) and potassium chloride
(99.999%, Aldrich) were used to make buffered solutions (50 mM PIPES,
100 mM KCl, pH 7.0) with Millipore water. Nitric oxide was purchased
from Airgas and was purified as described previously.61 Nitrogen dioxide
(NO2) was purchased from Aldrich and purified by condensation into a
gas bulb at ꢀ78 °C followed by exposure to vacuum to remove higher
boiling impurities. SNAP, sodium peroxynitrite, sodium nitrite, and Angeli’s
salt (Na2N2O3) were purchased from Cayman Chemical and stored
at ꢀ80 °C prior to use. Nitric oxide and NO2, as well as 1.0 mM
solutions of other reactive oxygen and nitrogen species (RONS), were
introduced into buffered solutions via gastight syringes. Copper(II)
chloride dihydrate (99+%, Alfa Aesar) was used to prepare 1.0 mM
CuCl2 stock solutions with Millipore water. Stock solutions of the
fluorescent probes were prepared in DMSO and stored in aliquots
at ꢀ80 °C until immediately prior to use. UVꢀvisible spectra were
acquired on a Cary 50-Bio or a Cary 1E spectrometer. Acquisitions were
made at25.00( 0.05 °C. Fluorescence spectra were obtainedona Quanta
Cell Culture and Imaging Materials and Methods. Raw
264.7 murine macrophages were obtained from ATCC and were cultured
in Dulbecco’s modified Eagle medium (DMEM, Cellgro, MediaTek, -
Inc.) supplemented with 10% fetal bovine serum (FBS, HyClone), 1%
penicillinꢀstreptomycin, 1% sodium pyruvate (Cellgro, MediaTek, Inc.),
and 1% MEM nonessential amino acids(Sigma). Forimaging studies, cells
grown to confluence were passed and plated into poly-D-lysine coated
plates (MatTek) containing 2 mL of DMEM. After incubation at 37 °C
with5% CO2 for atleast 5 h, the media wasremoved, thecells were washed
with 2 mL of PBS buffer, and 2 mL of fresh DMEM was added along with
the fluorescent probes and/or iNOS stimulants. Cells were incubated for
14ꢀ16 h prior to imaging. For all cell studies, the Cu(II) complexes of the
fluorescent probes were generated in situ by combining stock solutions of
CuCl2 and the dye. For all comparison studies, the plates were loaded with
identical volumes from the same cell stock solution to ensure that an equal
number of cells were passed to each plate.
For nitric oxide detection studies, NO production by iNOS was
induced in Raw 264.7 macrophages with 0.75 ng/mL of lipopolysac-
charide (LPS, Sigma) and495ꢀ4950 U/mLof interferon-gamma(IFN-γ,
BD Biosciences). The concentrations of LPS and INF-γ were optimized
using Cu2FL2E. Cells were then coincubated with either 2 μM CuSNFL1,
2 μM CuSNFL1Br, 4 μM CuSNFL1E, or 4 μM CuSNFL1EBr and
4.5 μM Hoechst 33258 (Sigma). Prior to imaging, cells were washed
with 2 mL of PBS and then bathed in 2 mL of warm PBS during imaging.
Fluorescence Microscopy. Images were acquired on a Zeiss
Axiovert 200 M inverted epifluorescence microscope equipped with an
EM-CCD digital camera (Hamamatsu) and an X-Cite 120 metal halide
lamp (EXFO). Differential interference contrast (DIC) and fluorescence
images were obtained using an oil immersion 63ꢁ objective lens, with
exposure time ranging from 250 ms to 1.75 s. Emission from the
seminaphthofluorescein-based probes was detected using a custom filter
set (Chroma) with a 500 nm excitation filter (HQ500/40ꢁ), a 530 nm
dichroic mirror (Q530LP), and a 550 nm long pass emission filter
(E550LP). The microscope was operated with Volocity software
Figure 5. Fluorescence response of CuSNFL1 and CuSNFL1Br to Zn(II).
The fluorescence response for NO is shown for comparison. Conditions: 10
μM CuCl2, 10 μM probe, 50 mM PIPES, 100 mM KCl, pH 7.0, 25 °C. The
Zn(II) concentration was adjusted with 10 mM or 1.0 mM ZnCl2.
Figure 6. Fluorescence imaging of endogenous NO produced in Raw 264.7 cells. For each set of images, the left image corresponds to treatment with
the fluorescent probe only. Images in the right panel correspond to cells treated with the fluorescent probes and stimulated with LPS and INF-γ. (Top)
DIC images. (Bottom) Fluorescence images. The fluorescence intensity of the uncorrected fluorescence images are shown below to compare the
fluorescence response in unstimulated and stimulated cells. [Probe] = 2 μM for CuSNFL1 and CuSFNL1Br, 4 μM for CuSNFL1E and CuSNFL1EBr.
[LPS] = 0.75 ng/mL, [INF-γ] = 495ꢀ4950 U/mL, incubation for 14ꢀ16 h. Scale bars = 23 μm.
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(Improvision) and images were analyzedwith either the Volocity software
or ImageJ.62 All fluorescent images were corrected for background.
Uncorrected images are shown in the Supporting Information (Figures
S15ꢀS18).
7 (6.2 mg, 49 μmol) were combined in 2 mL of anhydrous MeOH and
2 mL of anhydrous CH2Cl2 containing 4 Å molecular sieves. The solution
was protected from light and stirred for 1 h, after which the reaction
solution was cooled to 0 °C. Next, NaBH(OAc)3 (34 mg, 163 μmol) was
added in one portion. The solution was allowed to warm to room
temperature and stirred overnight. After filtration and removal of the
solvents under vacuum, the residual red solid was dissolved in a minimal
amount of MeOH and purified by preparative TLC eluting with 5%
MeOH in CH2Cl2 to afford SNFL1 (5.9 mg, 30% yield). Mp: 185ꢀ187
(dec). 1H NMR (400 MHz, 10:1 CD2Cl2:MeOH-d4): δ 8.38 (d, J = 9.2,
1H, ArH), 8.02 (d, J = 6.8 Hz, 1H, ArH), 7.96 (d, J = 8.4, 1H, ArH), 7.62
(m, 2H, ArH), 7.28 (overlapping m, 3H, ArH), 7.13 (overlapping m, 5H,
ArH), 6.69 (d, J = 8.4, 2H, ArH), 6.62 (d, J = 7.2, 1H, ArH), 5.02 (s, 2H,
CH2), 2.54 (s, 3H, CH3). HRMS ([M ꢀ H]ꢀ) calcd m/z 551.1612,
found m/z 551.1608.
2-(11-(((6-(2-Ethoxy-2-oxoethoxy)-2-methylquinolin-8-yl)amino)-
methyl)-10-hydroxy-3-oxo-3H-benzo[c]xanthen-7-yl)benzoic Acid,
SNFL1E. The seminaphthofluorescein aldehyde 5 (27.0 mg, 65.7 μmol)
and aminoquinaldine ester 8 (22.2 mg, 85.7 μmol) were combined in
2 mL of anhydrous MeOH and 2 mL of anhydrous CH2Cl2 containing
4 Å molecular sieves. The solution was protected from light and stirred
for 2 h, after which the reaction solution was cooled to 0 °C. After
cooling, NaBH(OAc)3 (25.1 mg, 118 μmol) was added in one portion.
The solution was allowed to warm to room temperature and stirred for 3
h. After filtration and removal of the solvents under vacuum, the red
residual solid was dissolved in a minimal amount of MeOH and purified
by preparative TLC eluting with 8% MeOH in CH2Cl2 to yield SNFL1E
(8.9 mg, 16% yield). Mp: 174ꢀ177 °C (dec). 1H NMR (400 MHz, 10:1
CD2Cl2:MeOH-d4): δ 8.45 (d, J = 8.8 Hz, 1H, ArH), 8.03 (bs, 1H, ArH),
7.81 (d, J = 8.0 Hz, 1H, ArH), 7.61 (bs, 2H, ArH), 7.27 (d, J = 8.8 Hz, 1H,
ArH), 7.17 (m, 3H, ArH), 7.13 (s, 1H, ArH), 6.80 (overlapping m, 3H,
ArH), 6.28 (d, J = 8.8 Hz, 1H, ArH), 6.29 (s, 1H, ArH), 4.90 (s, 2H,
CH2), 4.66 (s, 2H, CH2), 4.22 (q, J = 7.2 Hz, 2H, CH2), 2.44 (s, 3H,
CH3), 1.26 (t, J = 4.8 Hz, 3H, CH3). HRMS ([M ꢀ H]ꢀ): calcd m/z
653.1929, found m/z 653.1936.
2-(8-Bromo-10-hydroxy-11-(((2-methylquinolin-8-yl)amino)
methyl)-3-oxo-3H-benzo[c]xanthen-7-yl)benzoic acid, SNFL1Br. The
brominated seminaphthofluorescein aldehyde 6 (16.1 mg, 39.2 μmol)
and 2-methylquinolin-8-amine 7 (6.8 mg, 43 μmol) were combined in
2 mL of anhydrous MeOH and 3 mL of anhydrous CH2Cl2 containing
4 Å molecular sieves. The solution was protected from light and stirred
for 3 h, after which the reaction solution was cooled to 0 °C. After
cooling, NaBH(OAc)3 (16 mg, 78 μmol) was added in one portion. The
solution was allowed to warm to room temperature and stirred over-
night. After filtration and removal of the solvents under vacuum, the red
residual solid was dissolved in a minimal amount of MeOH and purified
by preparative TLC eluting with 10% MeOH in CH2Cl2 to yield
SNFL1Br (9.3 mg, 41% yield). Mp: 179ꢀ181 °C (dec). 1H NMR
(400 MHz, 10:1 CD2Cl2:MeOH-d4): δ 8.43 (d, J = 8.4, 1H, ArH), 8.06
(bs, 1H, ArH), 7.95 (d, J = 8.4, 1H, ArH), 7.66 (bs, 2H, ArH), 7.45
(s, 1H, ArH), 7.32 (t, J = 6.4, 1H, ArH), 7.18 (overlapping m, 4H, ArH),
7.09 (d, J = 8.4, 1H, ArH), 7.06 (s, 1H, ArH), 6.73 (d, J = 8.4, 1H, ArH),
6.64 (d, J = 8.4, 1H, ArH), 4.96 (s, 2H, CH2), 2.50 (s, 3H, CH3). HRMS
([M + H]+): calcd m/z 631.0863, found m/z 631.0841.
2-(8-Bromo-11-(((6-(2-ethoxy-2-oxoethoxy)-2-methylquinolin-8-yl)-
amino)methyl)-10-hydroxy-3-oxo-3H-benzo[c]xanthen-7-yl)benzoic Acid,
SFNL1EBr. The brominated seminaphthofluorescein aldehyde 6 (13.1 mg,
31.8 μmol) and aminoquinaldine ester 8 (10.4 mg, 39.4 μmol) were
combined in 1 mL of anhydrous MeOH and 1 mL of anhydrous CH2Cl2
containing 4 Å molecularsieves. The solution wasprotectedfrom light and
stirred for 2 h, after which the reaction solution was cooled to 0 °C. After
cooling, NaBH(OAc)3 (13.5 mg, 63.6 μmol) was added in one portion. The
reaction mixture was allowed to warm to room temperature and stirred for
2 h, after which it was filtered, and the solvents were removed under vacuum.
X-ray Data Collection and Structure Solution Refinement.
Crystals of 6 of suitable quality for X-ray diffraction were grown by
diffusion of hexanes into a CHCl3 solution of 6. A single crystal was
mounted in Paratone N oil using a 30 μm aperture MiTeGen Micro-
Mounts (Ithaca, NY) loop and frozen under a nitrogen cold stream
maintained by a KRYO-FLEX low-temperature apparatus set to 100 K.
Data were collected on a Bruker SMART APEX CCD X-ray diffract-
ometer with Mo KR radiation (λ = 0.710 73 Å) controlled by the APEX2
software package (v 2010).63 Data reduction was performed with
SAINT and empirical absorption corrections were applied with
SADABS.64 The structures were solved by direct methods with refine-
ment by full-matrix least-squares based on F2 using SHELXTL-97.65,66
All non-hydrogen atoms were located and their positions refined aniso-
tropically. Hydrogen atoms were assigned to idealized positions and given
thermal parameters equal to 1.2 times the thermal parameters of the
atoms to which they were attached.
Syntheses. 8-Bromo-11-(bromomethyl)-30-oxo-30H-spiro[ben-
zo[c]xanthene-7,10-isobenzofuran]-3,10-diyl Dibenzoate, 4. A mix-
ture of dibenzoate 1 (11.9 g, 19.7 mmol), 1,3-dibromo-5,5-dimethyl
hydantoin 2 (12.7 g, 44.4 mmol), VAZO 88 (660 mg, 2.71 mmol),
and glacial acetic acid (420 μL, 7.3 mmol) was added to 300 mL of
chlorobenzene and heated at 60 °C for 7 days. The resultant solution
was washed with hot water and dried with MgSO4, and the solvents were
removed under vacuum. The resultant solid was recrystallized from a
minimal amount of toluene and ethanol (∼10:1) to yield the desired
product (13.4 g, 89% yield). Note: If the bromination does not go to
completion, excess VAZO 88 or 2 can be added during the course of
the reaction. Mp: 171ꢀ173 °C (dec). 1H NMR (400 MHz, CDCl3): δ
8.72 (d, J = 8.8, 1H, ArH), 8.24 (d, J = 7.2, 4H, ArH), 8.06 (m, 2H,
ArH), 7.67 (m, 5H, ArH), 7.52 (m, 4H, ArH), 7.20 (d, J = 8.0, 1H,
ArH), 7.11 (s, 1H, ArH), 7.06 (d, J = 8.8, 1H, ArH), 6.91 (d, J = 8.4, 1H,
ArH) 4.88 (m, 2H, CH2). 13C{1H} NMR (100 MHz, CDCl3): δ
169.2, 165.2, 164.4, 152.9, 152.0, 149.4, 146.3, 135.5, 134.5, 134.2,
130.7, 130.6, 130.5, 129.24, 129.17, 129.10, 128.92, 128.7, 128.1,
126.1, 125.7, 124.7, 124.4, 123.7, 123.1, 119.35, 119.30, 119.22,
117.16, 117.05, 113.51, 81.7, 20.5. HRMS ([M + H]+): calcd m/z
760.9805, found m/z 760.9827. Anal. Calcd for C39H22Br2O7EtOH:
C, 60.91; H, 3.49. Found: C, 60.81; H, 3.21.
2-(11-Formyl-10-hydroxy-8-bromo-3-oxo-3H-benzo[c]xanthen-
7-yl)benzoic Acid, 6. The seminaphthofluorescein bromide 4 (2.0 g, 2.9
mmol) was combined with NaHCO3 (2.0 g, 24 mmol) in 70 mL of
DMSO and heated to 140 °C for 3 h. The reaction mixture was cooled to
room temperature, poured into 350 mL of 4 M HCl, and stirred for 30
min. The solution was filtered, washed with water, and dried under
vacuum. The resultant crude product was purified using two SiO2
columns. The first column was eluted with 10% MeOH in CH2Cl2.
The second column was eluted with 2% MeOH in CH2Cl2 to yield the
pure aldehyde 6 (59 mg, 5% yield). Mp: >300 °C. 1H NMR (400 MHz,
10:1 CD2Cl2:MeOD-d4): δ 8.35 (d, J = 9.2, 1H, ArH), 8.06 (d, J = 7.6,
1H, ArH), 7.72 (m, 2H, ArH), 7.45 (s, 1H, ArH), 7.29 (d, J = 9.2, 1H,
ArH), 7.17 (d, J = 7.6, 1H, ArH), 6.96 (s, 1H, ArH), 6.93 (d, J = 9.2, 1H,
ArH), 6.66 (d, J = 9.2, 1H, ArH). 13C{1H} NMR (100 MHz, 10:1
CD2Cl2:d4-MeOD): δ 193.5, 170.2, 164.8, 159.3, 153.1, 153.0, 146.5,
137.5, 136.5, 135.5, 131.1, 127.8, 126.9, 125.9, 124.8, 124.6, 120.4, 119.3,
116.8, 114.8, 111.3, 111.3, 111.1, 109.8, 82.8. HRMS ([M + H]+): calcd
m/z 488.9968, found m/z 488.9954. Anal. Calcd for C25H13BrO6
MeOH: C, 59.90; H, 3.29; Found: C, 60.13; H, 3.66.
3
2-(10-Hydroxy-11-(((2-methylquinolin-8-yl)amino)methyl)-3-oxo-
3H-benzo[c]xanthen-7-yl)benzoic Acid, SNFL1. The seminaphthofluor-
escein aldehyde 5 (13.4 mg, 32.7 μmol) and 2-methylquinolin-8-amine
9390
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Inorganic Chemistry
ARTICLE
The red residual solid was dissolved in a minimal amount of MeOH and
purified by preparative TLC eluting with 6% MeOH in CH2Cl2 to yield
SNFL1EBr (12 mg, 58% yield). Mp: 169ꢀ171 °C (dec). 1H NMR (400
MHz, MeOH-d4): δ 8.46 (d, J = 8.8, 1H, ArH), 8.05 (dd, J = 6.4, 2.4, 1H,
ArH), 7.80 (d, J = 8.4, 1H, ArH), 7.70 (m, 2H, ArH), 7.44 (d, J = 2.0, 1H,
ArH), 7.19(overlappingm, 2H, ArH), 7.12(d,J=8.4,1H, ArH), 6.49(s, 1H,
ArH), 6.87 (d, J = 1.6, 1H, ArH), 6.69 (s, 2H, ArH), 6.35 (d, J = 2.4, 1H,
ArH), 4.68 (s, 2H, CH2), 4.62 (bs, 2H, CH2), 4.22 (q, J = 6.8 Hz, 2H, CH2),
2.36 (s, 3H, CH3), 1.25(t, J= 6.8, 3H, CH3). HRMS ([M +H]+): calcd m/z
733.1180, found m/z 733.1177.
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S
Supporting Information. X-ray crystallographic data of
b
compound 6 in CIF format, excitation/emission plots for all
probes, selectivity plotsanddata forvarious excitation wavelengths,
pH profiles, raw cell images, cell washing studies, NMR spectra.
This material is available free of charge via the Internet at http://
pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: lippard@mit.edu.
’ ACKNOWLEDGMENT
This work was supported the National Science Foundation
(CHE-0611944 to SJL) and the National Institutes of Health
(K99GM092970 to MDP). Instrumentation in the MIT DCIF
is maintained with funding from NIH grant 1S10RR13886-01.
The authors thank Daniela Buccella for helpful discussions and
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