Triggered dye release via photodissociation of nitric oxide from designed ruthenium nitrosyls: Turn-ON fluorescence signaling of nitric oxide delivery

Article abstract of DOI:10.1021/ic201242d

Two new fluorescein-tethered nitrosyls derived from designed tetradentate ligands with carboxamido-N donors have been synthesized and characterized by spectroscopic techniques. These two diamagnetic {Ru-NO}⁶ nitrosyls, namely, [(Me₂b

Full text of DOI:10.1021/ic201242d

ARTICLE
pubs.acs.org/IC
Triggered Dye Release via Photodissociation of Nitric Oxide from
Designed Ruthenium Nitrosyls: Turn-ON Fluorescence Signaling of
Nitric Oxide Delivery
Nicole L. Fry, Julia Wei, and Pradip K. Mascharak*
Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, United States
S Supporting Information
b
ABSTRACT: Two new fluorescein-tethered nitrosyls derived
from designed tetradentate ligands with carboxamido-N donors
have been synthesized and characterized by spectroscopic
techniques. These two diamagnetic {Ru-NO}6 nitrosyls, namely,
[(Me2bpb)Ru(NO)(FlEt)] (1-FlEt, Me2bpb = 1,2-bis(pyridine-
2-carboxamido)5-dimethylbenzene, FlEt = fluorescein ethyl
ester) and [((OMe)2IQ1)Ru(NO)(FlEt)] (2-FlEt, (OMe)2-
IQ1 = 1,2-bis(isoquinoline-1-carboxamido)-4,5-dimethoxybenzene),
display NO stretching frequencies (νNO) at 1846 and 1832 cmꢀ1
in addition to their FlEt carbonyl stretching frequencies (νCO) at
1715 and 1712 cmꢀ1, respectively. Coordination of the dye ligand
enhances the absorptivity and NO photolability of these two
nitrosyls in the visible region (450ꢀ600 nm) of light. Exposure
to visible light promotes rapid loss of NO from both {Ru-NO}6 nitrosyls to generate Ru(III) photoproducts in dry aprotic solvents, such as
MeCN and DMF. The FlEtꢀ moiety remains bound to the paramagnetic Ru(III) center in such cases, and hence, the photoproducts
exhibit very weak fluorescence from the dye unit. In the presence of water, the Ru(III) photoproducts undergo further aquation and loss of
the FlEtꢀ moiety via protonation. These steps lead to turn-ON fluorescence (from the free FlEt unit) and provide a visual signal of the NO
photorelease from 1-FlEt and 2-FlEt in aqueous media.
’ INTRODUCTION
media compared to iron and manganese nitrosyls.16 However,
ruthenium nitrosyls with simple ligands (such as NH3 and
Cl)17,18 release NO only upon exposure to UV light, which itself
could also harm the biological target. Careful design of more
complex ligand frames has recently allowed one to overcome this
obstacle.19 In our synthetic strategy, beginning with the tetra-
dentate dicarboxamide ligand frame (H2bpb),20 addition of
electron-donating substituents on the phenylenediamine ring
(H2Me2bpb20 and H2(OMe)2bpb21) and exchange of the pyr-
idine units for quinoline moieties (H2Me2bQb,20 H2(OMe)2bQb,22
H2(OMe)IQ121) have so far resulted in ligands that promote
visible light absorption by the corresponding ruthenium nitrosyls.
For example, [(bpb)Ru(NO)(Cl)]20 releases NO upon exposure
to UV light (absorption maximum, λmax = 395 nm), whereas
[((OMe)2bQb)Ru(NO)(Cl)]22 exhibits moderate NO release
when exposed to visible light (λmax = 500 nm). To further
increase the efficiency of NO release with visible light, we
have employed a directly coordinated dye choromophore
to improve the extent of visible light absorption. Indeed,
the quantum yield at 500 nm (ϕ500) of NO release is in-
creased from 0.0008 for [(Me2bpb)Ru(NO)(Cl)] to 0.052 for
In recent years, the role of nitric oxide (NO) in biological
systems has been elucidated in detail.1ꢀ4 It is now known that
this small gaseous signaling molecule can greatly affect its physio-
logical response depending on its concentration. For example,
nanomolar concentrations of NO affect vasodilatation of smooth
muscles1,5 and neurotransmission in the brain.2 In contrast,
elevated NO concentrations in the micromolar range are em-
ployed by macrophages to eliminate pathogens.6,7 In addition,
high NO concentrations can lead to apoptosis (programmed
cell death).8ꢀ10 This latter finding suggests that malignant
cells can be destroyed via exposure to a high flux of NO. Such
an expectation has been realized in recent studies where
cancers of different grades and origins have been eradicated
via NO-induced apoptosis.8ꢀ10 These results have prompted
intense research activity in the area of development of
designed NO donors for selective delivery of NO to malignant
locales.11
Certain transition metals (Fe,12 Mn,13 and Ru14) can tightly
bind NO, forming metal nitrosyls that release NO upon exposure
to specific wavelengths of light. Such metal nitrosyls could
provide a way to deliver NO to selected sites as a new type of
photodynamic therapy (PDT).15 Ruthenium nitrosyls are pre-
ferable for biological use due to their inherent stability in aqueous
Received: June 9, 2011
Published: August 04, 2011
r
2011 American Chemical Society
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Inorganic Chemistry
ARTICLE
[(Me2bpb)Ru(NO)(Resf)] (1-Resf), in which the dye resorufin
(Resf) is bound to the Ru center.22
’ EXPERIMENTAL SECTION
Materials and General Procedures. NO gas was purchased
from Spectra Gases Inc. and was purified by passing through a long KOH
column prior to use. RuCl3 xH2O (Aldrich Chemical Co.) was treated
3
several times with concentrated HCl to prepare the starting metal salt,
RuCl3 3H2O. Fluorescein and AgBF4 were purchased from Fluka and
3
Alfa-Aesar, respectively. All solvents were dried by standard techniques
and distilled prior to use. The starting complexes [(Me2bpb)-
Ru(NO)(Cl)]20 and [((OMe)2IQ1)Ru(NO)(Cl)]21 were synthesized
by following procedures reported by us previously. The published
procedure for fluorescein ethyl ester25 was modified to obtain high
yields of the pure dye. All other chemicals were purchased from Aldrich
Chemical Co. and used without further purification.
Free resorufin is also a very efficient fluorophore that exhibits
red fluorescence upon light exposure. Although the fluorescence
of the free dye is partly quenched when Resf is directly attached
to the Ru center of the {RuNO}6 nitrosyls, the residual fluores-
cence of 1-Resf is enough to visualize the complex in cellular
matrixes.22 This allowed us to track the NO donor within the
biological targets. To extend this concept further and develop
new trackable NO donors, we chose fluorescein as the next
chromophore. Fluorescein is a green fluorescent dye commonly
used for biological studies.23,24 Ford and co-workers have recently
shown that attachment of a fluorescein dye derivative provides
fluorescence and moderate enhancement of the NO photolability of
a iron sulfur nitrosyl compound (Roussin’s red salt).25 However, the
fluorescein dye in this compound was attached to bridging S atom(s)
through a (CH2)2 linker. We were interested to see how the direct
attachment of fluorescein to the Ru center of our designed ruthenium
nitrosyls would affect the fluorescence and NO photolability of the
resulting nitrosyls upon exposure to 500 nm light. In this account, we
report the syntheses and spectroscopic properties of two new
{RuNO}6 nitrosyls with ligated fluorescein ethyl ester dye (FlEt),
namely, [(Me2bpb)Ru(NO)(FlEt)] (1-FlEt, Me2bpb = 1,2-bis-
(pyridine-2-carboxamido)-4,5-dimethylbenzene) and [((OMe)2-
IQ1)Ru(NO)(FlEt)] (2-FlEt, (OMe)2IQ1 = 1,2-bis(isoquinoline-
1-carboxamido)-4,5-dimethoxybenzene). Investigation of the
fluorescent properties and NO photolability of these two
complexes reveal that both nitrosyls exhibit turn-ON fluores-
cent signals triggered by photodissociation of NO upon exposure
to 500 nm light.
Syntheses of Compounds. Fluorescein Ethyl Ester (FlEt). A
slurry of fluorescein (1.00 g, 3.01 mmol) in 150 mL of EtOH was treated
with 7 mL of sulfuric acid and heated to reflux temperature for 20 h in a
500 mL round-bottom flask covered with aluminum foil. EtOH was then
removed via rotary evaporation, leaving a bright yellow oil. The product
was extracted into dichloromethane (DCM) via liquidꢀliquid extraction
using DCM and water, and the DCM layer was washed with sodium
bicarbonate (2.50 g, 30.00 mmol) dissolved in 50 mL of water. Finally,
DCM was removed via rotary evaporation, leaving a red-orange solid.
Yield: 0.97 g (90%). Selected IR frequencies (KBr disk, in cmꢀ1): 1718
(m), 1640 (m), 1595 (vs), 1495 (s), 1460 (vs), 1384 (s), 1263 (vs), 1205
1
(s), 1107 (s). H NMR in CDCl3, δ from TMS: 8.72 (d, 1H), 7.74
(t, 1H), 7.69 (t, 1H), 7.33 (d, 1H), 6.99 (d, 2H), 6.89 (s, 2H), 6.81 (d,
2H), 4.01 (q, 2H), 0.92 (t, 3H).
[(Me2bpb)Ru(NO)(FlEt)] (1-FlEt). A solution of 0.150 g of
[(Me2bpb)Ru(NO)(Cl)] (0.294 mmol) was prepared in 20 mL of
MeCN and treated with AgBF4 (0.057 g, 0.294 mmol). A separate
solution of 0.106 g of FlEt (0.294 mmol) in 20 mL of MeCN was treated
with NaH (0.007 g, 0.294 mmol). Both solutions were then heated to
reflux temperature, and the FlEt solution was slowly added to the
[(Me2bpb)Ru(NO)(Cl)] solution with stirring. Heating of the mixture
(with constant stirring) was continued at reflux temperature for 5 h.
Next, the bright red solution was cooled to ꢀ20 °C for 1 h to allow full
precipitation of impurities, which were filtered off using a Celite pad.
The filtrate was condensed to half the original volume, 5 mL of Et2O was
added, and then it was stored at ꢀ20 °C for 48 h. The red precipitate was
filtered, washed several times with Et2O, and dried in vacuo. It was finally
recrystallized from CHCl3. Yield: 0.100 g (41%). Anal. Calcd for
C42H31N5O8Ru (1-FlEt): C, 60.43; H, 3.74; N, 8.39. Found: C,
60.35; H, 3.49; N, 8.44. Selected IR frequencies (KBr disk, in cmꢀ1):
1846 (s), 1715 (m), 1635 (s), 1578 (vs), 1473 (s), 1379 (m), 1273 (s),
1206 (m), 1104 (m). 1H NMR in CDCl3, δ from TMS: 8.83 (d, 2H), 8.39
(s, 2H), 3.32 (d, 1H), 8.28 (d, 1H), 8.24 (t, 1H), 8.21 (t, 1H), 8.16 (d, 1H),
7.79 (t, 1H), 7.76 (t, 1H), 7.64 (t, 1H), 7.60 (t, 1H), 7.17 (d, 1H), 6.77
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ARTICLE
(d, 1H), 6.49 (d, 1H), 6.39 (d, 1H), 6.30 (s, 1H), 5.81 (d, 1H), 5.74 (s, 1H),
3.98 (q, 1H), 3.89 (q ,1H), 2.27 (s, 6H), 0.84 (t, 3H). ESI-MS: m/z 836
(M+). UVꢀvis in MeCN, λmax in nm (ε in Mꢀ1 cmꢀ1): 270 (25 500), 375
(11 000), 450 sh (18 500), 475 (24 000), 510 sh (20 100).
’ RESULTS AND DISCUSSION
Synthesis. To synthesize a fluorescein-bound ruthenium
nitrosyl, the structure of the dye molecule had to be considered.
For example, fluorescein has a phenolato-O as well as a carbox-
ylato-O donor center, both of which are capable of binding to the
metal center. Indeed, initial attempts to combine fluorescein with
our designed ruthenium nitrosyls resulted in a mixture of
products. To circumvent this problem, we decided to protect
one of the metal binding sites of the fluorescein dye. Simple treat-
ment of fluorescein with concentrated sulfuric acid in ethanol
resulted in conversion of the carboxylic acid into an ethyl ester,
thus leaving only the phenolato-O donor available for metal
binding. Removal of the chloride from [(Me2bpb)Ru(NO)(Cl)]
(1-Cl) with AgBF4 in MeCN allowed the fluorescein ethyl ester
(FlEt, deprotonated with NaH) to bind the Ru center, afford-
ing [(Me2bpb)Ru(NO)(FlEt)] (1-FlEt) in good yield. The
(Me2bpb)2ꢀ ligand frame was strategically chosen for its planarity
when bound to the ruthenium center.21 This allows ample room
for binding of the bulky FlEt dye trans to NO. Similarly, we also
selected the other planar ((OMe)2IQ1)2ꢀ ligand frame to isolate
[((OMe)2IQ1)Ru(NO)(FlEt)] (2-FlEt) from [((OMe)2IQ1)-
Ru(NO)(Cl)] (2-Cl) using the same synthetic procedure
mentioned above.
[((OMe)2IQ1)Ru(NO)(FlEt)] (2-FlEt). A batch of [((OMe)2IQ1)-
Ru(NO)(Cl)] (0.150 g, 0.233 mmol) was treated with AgBF4 (0.045
g, 0.233 mmol) in 15 mL of MeCN and heated to reflux temperature.
Meanwhile, a slurry of FlEt (0.084 g, 0.233 mmol) was treated with
1 equiv of NaH (0.006 g, 0.233 mmol) in MeCN and also brought to
reflux temperature. The hot FlEt solution was then added to the
[((OMe)2IQ1)Ru(NO)(Cl)] solution, and the mixture was kept at
reflux temperature for 8 h. Over the course of the reaction, a red-orange
precipitate separated out. The precipitate was collected by filtration and
recrystallized from CHCl3/pentane. Yield: 0.172 g (76%). Anal. Calcd
for C50H35N5O10Ru (2-FlEt): C, 62.11; H, 3.65; N, 7.24. Found: C,
62.17; H, 3.50; N, 7.42. Selected IR frequencies (KBr disk, in cmꢀ1):
1832 (m), 1712 (w), 1617 (vs), 1579 (vs), 1496 (s), 1332 (m), 1287 (s),
1213 (w), 1097 (s). 1H NMR in CDCl3, δ from TMS: 10.339 (d, 1H),
10.193 (d, 1H), 8.790 (dd, 2H), 8.521 (s, 1H), 8.482 (s, 1H), 8.141 (m,
3H), 7.996 (tt, 3H), 7.891 (dt, 2H), 7.809 (t, 1H), 7.596 (dt, 2H), 7.054 (d,
1H), 6.703 (d, 1H), 6.440 (d, 1H), 6.293(d, 1H), 6.057 (s, 1H), 5.875 (d,
1H), 5.363 (s, 1H), 4.012 (s, 3H), 3.994 (s, 3H), 3.885 (q, 1H), 3.796 (q,
1H), 0.701 (t, 3H). ESI-MS: m/z 968 (M+). UVꢀvis in MeCN, λmax in nm
(ε in Mꢀ1 cmꢀ1): 280 (28 700), 320 sh (17 000), 365 sh (10 200), 450 sh
(21 00), 475 (28 000), 510 sh (22 700).
Physical Measurements. The 1H NMR spectra were recorded at
298 K on a Varian Inova 500 MHz instrument. A PerkinElmer
Spectrum-One FT-IR spectrometer was used to monitor the IR spectra
of the complexes. The electronic absorption spectra were obtained with
a scanning Cary 50 spectrophotometer (Varian Associates). Fluores-
cence spectra were recorded with a PerkinElmer LS50B fluorescence/
luminescence spectrometer. X-band electron paramagnetic resonance
(EPR) spectra were obtained with a Bruker ELEXSYS 500 spectrometer
at 125 K. Electrospray ionization mass spectrometry (ESI-MS) was
carried out on a Waters Micromass ZMD mass spectrometer. Release of
NO upon illumination in aqueous solution was monitored by using the
inNO Nitric Oxide Monitoring System (Innovative Instruments Inc.)
fitted with an ami-NO 2008 electrode. The NO amperograms were
recorded using stirred solutions contained in open vials.
Photolysis Experiments. The quantum yield (ϕ) values of NO
release were obtained using a tunable Apex Illuminator (150 W xenon
lamp) equipped with a Cornerstone 130 1/8 M monochromator
(measured intensity of ∼10 mW). Actinochrome N (475/610) was
used to as the standard for the quantum yield values calculated at 500 nm
(ϕ500).26 Solutions of 1-FlEt and 2-FlEt were prepared and placed in 2 ꢁ
10 mm quartz cuvettes, 1 cm away from the light source. All solutions
were prepared to ensure sufficient absorbance (>90%) at the irradiation
wavelength, and changes in electronic spectrum at 750 and 700 nm for 1-
FlEt and 2-FlEt, respectively (<10% photolysis), were used to determine
the extent of photorelease of NO.
Fluorescence Experiments. Fluorescence spectra were recorded
with a PerkinElmer LS50B fluorescence/luminescence spectrometer. All
samples were prepared in four-sided 1 cm ꢁ 1 cm quartz cuvettes such
that the absorbance was <0.1 at the excitation wavelength. Fluorescence
quantum yields were determined relative to fluorescein in 0.1 N NaOH
(ϕ = 0.95).27 The concentration of the reference was adjusted to match
the absorbance of the test sample at the excitation wavelength (480 nm).
The fluorescence intensity of the resulting fluorescence spectra was
integrated from 500 to 650 nm for comparison. Fluorescence turn-ON
measurements of 1-FlEt and 2-FlEt were obtained upon comparison of
samples kept in the dark or exposed to visible light (1 min intervals) from
an IL 410 Illumination System from Electro-FiberOptics Corp. (halogen
lamp) equipped with a λ g 465 nm cutoff filter (measured intensity =
300 mW).
Spectroscopic Properties. The structures of 1-FlEt and 2-
FlEt have been confirmed with the aid of 1H NMR and infrared
(IR) spectroscopy, and mass spectrometry (see the Experimental
Section). IR spectra of 1-FlEt and 2-FlEt (Supporting Informa-
tion, Figures S1 and S2) reveal the presence of NO and FlEt, as
evidenced by their NO stretching frequencies (νNO) at 1846 and
1832 cmꢀ1 in addition to their FlEt carbonyl stretching frequen-
cies (νCO) at 1715 and 1712 cmꢀ1, respectively. The greater
electron-donating ability of the (OMe)2IQ1 ligand frame causes
an increase in electron density in the π* level of the bound NO of
2-FlEt and is responsible for its lower νNO frequency.
1
Both complexes are diamagnetic and afford clean H NMR
spectra, as expected for {RuNO}6 nitrosyls. The integration and
number of the peaks observed in both 1H NMR spectra confirm
1
the presence of Ru-bound FlEt. For example, in the H NMR
spectrum of free FlEt, there are several overlapping aromatic
peaks that shift apart upon binding to the Ru center. Thus, none
of the 10 aromatic FlEt hydrogen peaks overlap with one another
in the 1H NMR spectra of 1-FlEt or 2-FlEt in CDCl3 (see the
Supporting Information, Figures S4 and S5). Similarly, several
peaks corresponding to the hydrogen atoms on the ligand frame
shift upon metal binding. In addition, there is no evidence of
either Cl-bound or free FlEt starting materials in either spectrum.
Electronic Absorption Spectra. Our pervious studies have
shown that the attachment of suitable dye chromophores en-
hances the visible light absorption of {RuNO}6 nitrosyls.22 In the
present work, we have utilized the fluorescein ethyl ester FlEt
(and not fluoroscein) as the light-harvesting chromophore. The
protection of the carboxylate group of fluoroscein (necessary for
specific binding to the Ru center of the nitrosyl), however, did not
eliminate the visible light absorption by the dye. In its deprotonated
form, FlEtꢀ has an intense absorption band at 504 nm (ε = 80 000,
in 50:50 MeCN/H2O). Interestingly, there is a 50 nm blue shift (to
454 nm) coupled with a reduction in intensity (ε = 20 000, in
MeCN/AcOH) of this absorption band when FlEt is in its
protonated state (FlEt-H, Figure 1). Similar changes in the intensity
of the dye band are also observed upon coordination of the dye to
the Ru center in 1-FlEt and 2-FlEt (Figure 2).
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The electronic absorbance spectra of 1-FlEt and 2-FlEt are
very similar, with the main absorption bands of each nitrosyl
centered at 475 nm (Figure 2). The extinction coefficient values
at 475 nm for these FlEt-bound {RuNO}6 nitrosyls are quite high
(ε = 24 000 and 28 000 Mꢀ1 cmꢀ1, respectively). In addition, the
general shapes of the absorption bands in both spectra are very
similar to that of FlEt-H, each containing three overlapping
peaks in the visible region. The high ε values of 1-FlEt and 2-FlEt
over this large range of wavelengths (400ꢀ550 nm) lead to a
significant amount of visible light absorption by these
dyeꢀnitrosyl conjugates. Indeed, the attachment of FlEt in both
nitrosyls has significantly enhanced the amount of visible light
absorption compared with the corresponding Cl-bound
{RuNO}6 nitrosyls (Figure 2). For example, the lowest-energy
absorbance band in the spectrum of [(Me2bpb)Ru(NO)(Cl)]
(1-Cl) has a λmax at 395 nm with an ε of 5300 Mꢀ1 cmꢀ1. There
is increased visible light absorption in the case of [((OMe)2-
IQ1)Ru(NO)(Cl)] (2-Cl) due to the replacement of the methyl
and pyridine substituents (in Me2bpb) with more electron-
donating methoxy groups and more conjugated quinoline do-
nors (in (OMe)2IQ1), respectively. Thus, 2-Cl has a λmax at
475 nm with an ε value of 8700 Mꢀ1 cmꢀ1. Similarly, the
increased visible light absorption due to the (OMe)2IQ1 ligand
frame also seems to cause an increase in ε value for 2-FlEt
compared with that of 1-FlEt.
such as MeCN, DMF, H2O, and PBS buffer, undergo similar
rapid changes in their electronic spectra (Figure 3 and Figures S6
and S7, Supporting Information). In addition, no significant
changes were observed in the spectra when such solutions were
kept in the dark for several hours. Given the known NO
photolability of similar {RuNO}6 nitrosyls,14,19 the light-induced
absorption changes can be linked to the photorelease of NO (and
FlEt, vide infra), which has been confirmed by measurements
with a NO-sensitive electrode (Figure 3, inset). Specifically, the
formation of a low-energy absorbance band (at 750 and 700 nm
in the case of 1-FlEt and 2-FlEt, respectively) is indicative of the
formation of a Ru(III) photoproduct upon NO release in both
aqueous and nonaqueous solvents. Such transitions have
previously been assigned as ligand-to-metal charge-transfer
(LMCT) bands for the Ru(III) photoproducts of structurally
related ruthenium nitrosyls.28 Monitoring the rate of increase of
these low-energy absorbance bands upon light exposure allows
determination of quantum yield values (ϕ) of NO release for
these {RuNO}6 nitrosyls. Comparison of the these quantum
yield values (measured in DMF for comparison with previously
synthesized nitrosyls) reveals an increased efficiency of NO
Photorelease of NO. Upon exposure to visible light (λ >
465 nm, 300 mW), solutions of 1-FlEt and 2-FlEt in solvents,
Figure 3. Changes in the electronic absorption spectrum upon photo-
lysis of 1-FlEt in MeCN following illumination with visible light. Inset:
NO amperogram of 1-FlEt in 50:50 MeCN/H2O upon illumination
with visible light for time periods (in seconds) as indicated.
Figure 1. Electronic absorption spectra of fluorescein ethyl ester (FlEt,
in 50:50 MeCN/H2O) and protonated FlEt-H (in MeCN/AcOH pH = 5).
Figure 2. Electronic absorption spectra of [(Me2bpb)Ru(NO)(Cl)] and [(Me2bpb)Ru(NO)(FlEt)] (left panel) and [((OMe)2IQ1)Ru(NO)(Cl)]
and [((OMe)2IQ1)Ru(NO)(Cl)] (right panel) in MeCN.
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Table 1. Summary of Absorption Parameters (λmax, ε) and Quantum Yield (O) Values of NO Release from Selected DyeꢀNitrosyl
Conjugates
complex
quantum yield ϕ (λirr, nm)
solvent
DMF
λmax, nm (ε, Mꢀ1 cmꢀ1
)
[(Me2bpb)Ru(NO)(Cl)] (1-Cl) a
[(Me2bpb)Ru(NO)(Resf)] (1-Resf)b
[(Me2bpb)Ru(NO)(FlEt)] (1-FlEt)
[((OMe)2IQ1)Ru(NO)(Cl)] (2-Cl)a
[((OMe)2IQ1)Ru(NO)(Resf)] (2-Resf)b
[((OMe)2IQ1)Ru(NO)(FlEt)] (2-FlEt)
Roussin’s salt ester (RSE)c
0.0008 ( 0.0002 (500)
0.052 ( 0.008 (500)
0.306 ( 0.01 (500)
395 (5300)
500 (11 920)
475 (24 000)
475 (8700)
DMF
DMF
0.035 ( 0.005 (500)
0.271 ( 0.008 (500)
0.173 ( 0.01 (500)
0.00019 ( 0.00005(546)
0.0036 ( 0.0005(436)
DMF
DMF
500 (31 000)
475 (28 000)
364 (8500)
DMF
CHCl3
MeCN/H2O
Fluor-RSEd
506 (72 200)
a Reference 21. b Reference 22. c Reference 29. d Reference 25.
Table 2. Summary of Absorption and Emission Parameters
(λex = 480 nm) and Fluorescence Quantum Yield (Ofl) Values
of Selected Compounds in 50:50 MeCN/H2O at pH 7
release for the FlEt-bound nitroyls (1 and 2) compared with their
parent Cl-bound nitrosyls (1-Cl and 2-Cl; see Table 1). For
example, exposure of 1-Cl to 500 nm light results in a very low
quantum yield (ϕ500) value of 0.0008, whereas a similar light
exposure of 1-FlEt leads to a significantly larger ϕ500 value of
0.30. Interestingly, 1-FlEt has a larger ϕ500 compared with that of
2-FlEt (ϕ500 = 0.17). The opposite is true in the case of the
corresponding Resf-tethered {Ru-NO}6 nitrosyls. For example,
the ϕ500 value of the Resf-bound nitrosyls [(OMe)2IQ1)Ru-
(Resf)] (2-Resf)21 is larger than that of [(Me2bpb)Ru(Resf)] (1-
Resf, Table 1).22 Comparison of the extinction coefficient (ε)
values at 500 nm of 1-Resf (ε = 11 920 Mꢀ1 cmꢀ1) and 2-Resf
(ε = 31 000 Mꢀ1 cmꢀ1) suggests that the increased absorption at
500 nm for 2-Resf (compared to 1-Resf) could account for its
larger ϕ500 value. However, the same is not true for the FlEt-
tethered nitrosyls. Despite similar extinction coefficient values,
1-FlEt (ε500 = 19 000 Mꢀ1 cmꢀ1) exhibits a higher ϕ500 value
than 2-FlEt (ε500 = 22 000 Mꢀ1 cmꢀ1). The latter observation
indicates that increased absorption of light cannot be the only
reason for the enhanced NO photorelease. The reason for the
accelerated NO release upon illumination, therefore, awaits more
experimental and theoretical investigation. Such investigation is
in progress at this time.
Fluorescence Turn-ON. Given the intense fluorescence of
free fluorescein, we were interested to see if 1-FlEt and 2-FlEt
also had fluorescent properties. Because the ethyl ester fluor-
escein derivative was used for these nitroyls, we first examined
the fluorescent properties of FlEt. The conversion of fluorescein
into FlEt results in a small decrease in its fluorescence quantum
yield (ϕfl = 0.93 and 0.77, respectively, Table 2).25 However,
upon direct coordination of FlEt to the {RuNO}6 center in both
1-FlEt and 2-FlEt, the fluorescence is almost completely quenched
(ϕfl = 0.009 and 0.017, respectively). Interestingly, there is a rapid
increase in the fluorescence of solutions of 1-FlEt and 2-FlEt in
50:50 MeCN/H2O upon exposure to light (NO photorelease). In
addition, the residual fluorescence observed for solutions of 1-FlEt
and 2-FlEt becomes completely quenched upon exposure to light
in very dry solvents (such as MeCN). It is thus evident that these
FlEt-bound {RuNO}6 nitrosyls are light-activated fluorescence
turn-ON agents only in the presence of water. Figure 4 shows the
increase in fluorescence (λex = 480 nm, λem = 526 nm) of 1-FlEt
and 2-FlEt (in 50:50 MeCN/H2O) observed after 1 min intervals
of visible light exposure. There is a 27-fold fluorescence turn-ON
for 1-FlEt after 5 min of light exposure. In the case of 2-FlEt, there
is a 5-fold fluorescence increase under similar conditions. These
findings follow a similar trend (1-FlEt > 2-FlEt) to that observed
absorbance
emission
fluorescence
compound
λmax
λmax
quantum yield ϕfl
fluorescein
FlEt
496 nm
504 nm
475 nm
475 nm
518 nm
526 nm
526 nm
526 nm
0.93 ( 0.02 a
0.77 ( 0.02 b
0.009 ( 0.002
0.017 ( 0.003
1-FlEt
2-FlEt
a Measured in aqueous solution at pH 11. b Measured in a 50:50 mixture
of MeCN/phosphate buffer at pH 7.4.
for the NO photolability of 1-FlEt and 2-FlEt. Thus, the extent of
fluorescence turn-ON correlates with the amount of light-triggered
NO release from these {RuNO}6 nitrosyls and indicates that the
two reactions are intimately connected. Also, the results clearly
demonstrate that both NO release and fluorescence turn-ON
occurs only upon exposure to light since neither reaction occurs
when the solutions of 1-FlEt and 2-FlEt (in aqueous or nonaqu-
eous media) are kept in the dark. Although overlap of the dye
bands (free and bound, in the range of 400ꢀ510 nm) and fluor-
escence bands (also in the same region) prevents one to quantita-
tively correlate the extent of such turn-ON with the light-triggered
NO release in the present case, the dyeꢀnitrosyl conjugates 1-FlEt
and 2-FlEt do provide turn-ON signals upon NO release that
could be effectively utilized in biological systems.
To understand the structural changes responsible for the
increase in fluorescence, we have employed 1H NMR spectros-
copy to monitor the fate of 1-FlEt and 2-FlEt upon light
exposure in wet CDCN3. When left in the dark for several hours,
there are no changes in the 1H NMR spectra of both nitrosyls.
However, exposure of the solutions of 1-FlEt and 2-FlEt to
visible light results in the appearance of new peaks consistent
with those of free FlEt (Figure 5 and Figure S8, Supporting
Information). The new dye peaks do not show significant line
broadening after initial light exposure due to the low concentra-
tion of the Ru(III) photoproduct in the solution. However, once
1
the samples have been fully photolyzed, the H NMR spectra
broaden considerably. In addition, a low-spin Ru(III) EPR signal
is observed for such solutions (vide infa). Thus, it becomes clear
that the increase in fluorescence observed upon light exposure in
water is due to the release of FlEt from the Ru(III) photoproduct.
We hypothesize that this release of FlEt arises due to the
formation of a thermodynamically stable HO-bound Ru(III)
photoproduct. When NO is bound, the Ru center is thought to
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Figure 4. Changes in the fluorescence emission spectra upon 1 min intervals of visible light illumination of 1-FlEt (left panel) and 2-FlEt (right panel) in
50:50 H2O/MeCN (λex = 480 nm). Samples with absorbance values of 0.09 at 480 nm were used.
Figure 5. Proton NMR spectra (9.1ꢀ5.5 ppm) of 1-FlEt in CD3CN at 298 K kept in the dark (bottom) and after light exposure (top). The red arrows
show new peaks of unbound FlEt dye.
have more low-spin Ru(II) character. However, once NO has
been released, the Ru center is converted into a low-spin Ru(III)
species that is more susceptible to aquation (eq 1). The high
Lewis acidity of the resulting water-bound Ru(III) complex
combined with the basic properties of FlEtꢀ may eventually lead
to the formation of the more stable [(L)Ru(III)(OH)(H2O)]
(L = Me2bpb or (OMe)2IQ1) photoproduct and FlEt-H (eq 2).
Indeed, changes in the absorption spectrum of 1-FlEt upon light
exposure in aqueous solvents (Figure S6, Supporting Infor-
mation) suggest that the free dye ultimately becomes protonated
because the intensity of the free dye band is similar to that of the
original dye-bound 1-FlEt absorption band (Figures 1 and 2). If
the free dye were fully deprotonated, the intensity of the band
would be expected to be much greater since it has a larger
extinction coefficient value.
release. The small residual fluorescence observed for 1-FlEt and
2-FlEt in such solvents is also quenched following the NO loss.
However, the resulting paramagnetic low-spin Ru(III) center of
the dye photoproducts [(L)Ru(III)(FlEt)(solv)] (L = Me2bpb
or (OMe)2IQ1) is responsible for the fluorescence quenching.
To confirm the 3+ oxidation state of ruthenium in the solvato
species generated after complete photolysis of the diamagnetic
{Ru-NO}6 nitrosyls 1-FlEt and 2-FlEt, EPR measurements were
performed. Photoproducts of 1-FlEt and 2-FlEt generated in dry
MeCN display axial EPR spectra (g values: 2.16 and 1.89 for
1-FlEt and 2.18 and 1.90 for 2-FlEt) typical of low-spin Ru(III).
In contrast, the spectra change when photolysis is done in the
presence of water (g values: 2.19 and 1.88 for 1-FlEt and 2.12 and
1.92 for 2-FlEt). Comparison of the EPR spectra of the photo-
product of 1-FlEt in aqueous media (Figure S8, Supporting
Information) with that of [(Me2bpb)Ru(NO)(OH)]22 after
photolysis under similar conditions confirms such a conclusion
(similar spectra with identical g values). Collectively, these results
support the hypothesis that the ultimate formation of the OH-
bound photoproduct [(L)Ru(III)(OH)(H2O)] (L = Me2bpb or
(OMe)2IQ1) drives the release of FlEt.
H2O
f
½ðLÞRuðNOÞðFlEtÞꢂ
s
NO• þ ½ðLÞRu3þðH2OÞðFlEtÞꢂþ
ð1Þ
H2O
f
½ðLÞRu3þðH2OÞðElEtÞꢂþ
s
½ðLÞRu3þðH2OÞðOHÞꢂ þ FlEt-H
Previously, we shown that {RuNO}6 nitrosyls with coordi-
nated Resf21,22 or dansyl30 dye chromophores display turn-OFF
fluorescence signal once NO is released. The decrease in
fluorescence has been attributed to the formation of dye-bound
Ru(III) photoproducts. The paramagnetic d5 Ru(III) center is a
very effective fluorescence quencher of the bound Resf and
ð2Þ
ðL ¼ Me2bpb orðOMeÞ2IQ 1Þ
In dry nonaqueous solvents, the FlEt dye in 1-FlEt and 2-FlEt
stays bound to the Ru(III) center upon light exposure and NO
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Scheme 1
dansyl-imidazole (Ds-im) dyes in these photoproducts. In con-
trast, 1-FlEt and 2-FlEt are the first examples of ruthenium
nitrosyls with a fluorescent turn-ON signal for NO release.
Although a Ru(III) photoproduct is also produced in this case,
the FlEt dye does not stay bound to the metal center in the
presence of water. It becomes apparent that FlEt has a lower
affinity for Ru(III) compared with that of Resf or Ds-im. Thus, as
NO leaves, generating a Ru(III) photoproduct, FlEt is released
and a fluorescence enhancement is noted instead of quenching
(Scheme 1, top).
and 2-FlEt (Figures S3, S4, and S5), changes in the electronic
absorption (Figures S6 and S7) and H NMR (Figure S8)
spectra upon photolysis of 2-FlEt, and EPR spectrum of the
photoproduct of 1-FlEt in 50:50 MeCN/H2O (Figure S9).
This material is available free of charge via the Internet at
http://pubs.acs.org.
1
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: pradip@chemistry.ucsc.edu.
Recently, there have been several reports of fluorescent turn-
ON sensors that signal the presence of NO.31 For example,
Lippard and co-workers have designed a Cu(II) fluorescein-
based NO sensor CuFL (FL = 2-{2-chloro-6-hydroxy-5-[(2-
methyl-quinolin-8-ylamino)-methyl]-3-oxo-3H-xanthen-9-yl}-
benzoic acid).32 The FL ligand is not fluorescent in its free or
Cu(II)-bound form. However, upon expose to NO, the Cu(II)
center of CuFl is reduced to Cu(I) with concomitant N-
nitrosylation of FL (FL-NO). The N-nitrosylation of the FL-NO
ligand results in its displacement from the Cu(I) center and an
increase in fluorescence (Scheme 1, bottom). Indeed, CuFL has
been successfully used to visualize NO generated from iNOS in
murine macrophage cells and from cNOS in SK-N-SH human
neuroblastoma cells.32 However, in the present case, our goal is to
deliver exogenous NO to biological targets. With 1-FlEt and 2-
FlEt, the release of NO (instead of NO binding) results in a
fluorescent turn-ON signal that could clearly indicate NO delivery.
This is a distinct advantage of the designed NO donors 1-FlEt and
2-FlEt. Studies on the utility of these nitrosyls in NO delivery to
biological targets are in progress in this laboratory.
’ ACKNOWLEDGMENT
Financial support from a grant from the National Science
Foundation (CHE-0957251) is gratefully acknowledged. N.L.F.
received partial support from a COR-SRG grant from UCSC.
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