Characterization and Biological Activity of a Hydrogen Sulfide-Releasing Red Light-Activated Ruthenium(II) Complex

Article abstract of DOI:10.1021/jacs.8b08695

Hydrogen sulfide (H₂S) is a biological gasotransmitter that has been employed for the treatment of ischemia-reperfusion injury. Despite its therapeutic value, the implementation of this gaseous molecule for this purpose has required H₂S-releasing prodrugs for effective intracellular delivery. The majority of these prodrugs, however, spontaneously release H₂S via uncontrolled hydrolysis. Here, we describe a Ru(II)-based H₂S-releasing agent that can be activated selectively by red light irradiation. This compound operates in living cells, increasing intracellular H₂S concentration only upon irradiation with red light. Furthermore, the red light irradiation of this compound protects H9c2 cardiomyoblasts from an in vitro model of ischemia-reperfusion injury. These results validate the use of red light-activated H₂S-releasing agents as valuable tools for studying the biology and therapeutic utility of this gasotransmitter.

Full text of DOI:10.1021/jacs.8b08695

Communication
pubs.acs.org/JACS
Cite This: J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
Characterization and Biological Activity of a Hydrogen Sulfide-
Releasing Red Light-Activated Ruthenium(II) Complex
Joshua J. Woods,†,§ Jian Cao,‡ Alexander R. Lippert,‡ and Justin J. Wilson*,§
†Robert F. Smith School for Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York 14853, United States
§Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, United States
‡Department of Chemistry, Southern Methodist University, Dallas, Texas 75275, United States
S
* Supporting Information
these systems require upconverting nanoparticles to mediate the
low-energy photoactivation process.21,23 Because red light
ABSTRACT: Hydrogen sulfide (H2S) is a biological
gasotransmitter that has been employed for the treatment
of ischemia-reperfusion injury. Despite its therapeutic
value, the implementation of this gaseous molecule for
this purpose has required H2S-releasing prodrugs for
effective intracellular delivery. The majority of these
prodrugs, however, spontaneously release H2S via
uncontrolled hydrolysis. Here, we describe a Ru(II)-
based H2S-releasing agent that can be activated selectively
by red light irradiation. This compound operates in living
cells, increasing intracellular H2S concentration only upon
irradiation with red light. Furthermore, the red light
irradiation of this compound protects H9c2 cardiomyo-
blasts from an in vitro model of ischemia-reperfusion
injury. These results validate the use of red light-activated
H2S-releasing agents as valuable tools for studying the
biology and therapeutic utility of this gasotransmitter.
effectively penetrates biological tissue and is nontoxic, the
development of small-molecule red light-activated H2S-releasing
agents would be particularly valuable for both studying the
biological roles of H2S and leveraging the therapeutic effects of
this gasotransmitter.39 In this report, we describe the first
prototype of this class of molecules and its implementation to
protect against in vitro ischemia-reperfusion injury.
Our strategy to develop such a red light-activated H2S-
releasing molecule invoked a combination of the established
H2S-releasing compound morpholin-4-ium 4-methoxyphenyl-
(morpholino)phosphinodithioate (GYY4137, Chart 1)40,41 and
Chart 1. GYY4137, and the Compounds [1]+ and [2]+ That
Were Explored in This Study
he biological role of the toxic gas hydrogen sulfide (H2S) as
T
a neuromodulator was first recognized in 1996.1 Since this
initial discovery, H2S has received significant attention because
of its therapeutic potential for treating inflammation,2
Parkinson’s disease,3 reproductive dysfunction,4 brain injury,5−9
diabetes,10 cancer,11 and ischemia-reperfusion (I/R) injury.12,13
The therapeutic implementation of H2S in its gaseous state,
however, is challenged by difficulties in administering bio-
logically relevant and beneficial concentrations while avoiding
problems associated with its known cytotoxicity, volatility, and
flammability.14−16 To circumvent this challenge, researchers
have developed synthetic donors that release H2S in response to
stimuli such as pH,17 external light,18−25 reactive oxygen
species,26 and enzymatic activity.26−32 Among these strategies,
light-activated H2S release has been recognized as a promising
tool for biomedical and therapeutic applications.18,21 Light-
activated prodrugs are exciting therapeutic candidates that allow
for localized and noninvasive treatment of serious medical
conditions, while circumventing toxic side effects that arise from
traditional chemotherapy.33−37
a photolabile ruthenium(II) scaffold [Ru(tpy)(biq)(L)]n+ (tpy
= 2,2′:6′-2″-terpyridine; biq = 2,2′-biquinoline, n = 1, 2). Ru(II)
compounds of this class possess low energy metal-to-ligand
charge transfer (MLCT) absorption bands that extend into the
red region of the visible spectrum. After population of the
1MLCT state by absorption of red light, efficient intersystem
crossing to a dissociative triplet ligand field (3LF) state ejects the
monodentate ligand L with high quantum yields.42−45
In aqueous or wet organic solvent, GYY4137 releases H2S
over a few hours.46 Sustained H2S release from GYY4137 has
been used therapeutically to treat inflammation,47 inhibit cancer
cell growth,48 and prevent ischemia-reperfusion injury.49,50 The
hydrolysis of GYY4137, however, occurs spontaneously in
solution, limiting spatiotemporal control of H2S release from
The majority of light-activated H2S-releasing agents require
UV light, which ineffectively penetrates biological tissue and can
give rise to toxic effects.38 Efforts to move photoactivation
wavelengths to more biologically useful regions of the visible
spectrum have resulted in several systems that can be triggered
with visible and near-infrared light.21−23 Except in one case,22
Received: August 13, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/jacs.8b08695
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Journal of the American Chemical Society
Communication
this molecule. We hypothesized that coordination of GYY4137
to the photoactive [Ru(tpy)(biq)(L)]n+ scaffold would inhibit
its spontaneous hydrolysis until it was released by irradiation
with red light. Thus, the complex [Ru(tpy)(biq)(GYY4137)]Cl
([1]Cl) (Chart 1) was developed as the first red light-activated
H2S-releasing molecule.
Table 1. Absorption Maxima (λmax), Molar Extinction
Coefficients (ε) at λmax, and Photosubstitution Quantum
Yields (Φ626) of [1]Cl and [2]Cl
Compound
λmax (nm)
ε (M−1 cm−1
)
Φ626 (%)
[1]Cl
[2]Cl
581
570
4050 140
4000 200
0.85 0.03
1.02 0.11
The direct reaction between [Ru(tpy)(biq)Cl]Cl and excess
GYY4137 in 50% aqueous acetone gave [1]Cl as the major
product in solution. Further purification by silica gel
chromatography (90/10 CH3CN/H2O) afforded pure [1]Cl
in 24% yield. When [Ru(tpy)(biq)Cl]PF6 was sequentially
treated with AgPF6, which removed the inner-sphere chloride as
insoluble AgCl, and excess GYY4137 in refluxing methanol, we
unexpectedly isolated [Ru(tpy)(biq)(GYYOMe)]Cl ([2]Cl),
where GYYOMe is O-methoxy 4-methoxyphenylphosphinodi-
thioate. Over the course of this reaction, the morpholine group
of GYY4137 was replaced by the methanol solvent giving rise to
the GYYOMe ligand (Chart 1).51
1MLCT absorption bands at λ = 581 nm ([1]Cl) and λ = 568 nm
([2]Cl), which both tail past 650 nm. Upon irradiation of the
complexes with 626 nm light (Figures S13 and S14, SI), the
1
UV−vis spectra evolve, resulting in a blue shift of the MLCT
band to a new maximum at 549 nm, characteristic of the
expected photoproduct [Ru(tpy)(biq)(OH2)]2+ (Figures 2 and
In addition to characterization by standard techniques, such
as NMR spectroscopy, mass spectrometry, and IR spectroscopy
(Figures S1−S12, Supporting Information, SI), [1]PF6 was also
characterized by single-crystal X-ray diffraction (Figure 1).
Figure 1. X-ray crystal structure of [1]PF6. Hydrogen atoms and the
PF6− counterion are omitted for clarity. Thermal ellipsoids are shown at
the 50% probability level.
Figure 2. Top: Changes in the electronic absorption spectrum of [1]Cl
in 100 mM MOPS (pH 7.4) under 626 nm light irradiation (photon
flux = 2.01 × 10−8 mol s−1, tirr = 15 min, T = 298 K). Bottom: Electronic
absorption spectrum of [1]Cl in 100 mM MOPS in the dark after 5 days
(T = 298 K).
Crystallographic parameters and relevant interatomic distances
and angles are listed in Tables S1 and S2 in the SI. The Ru(II)
center of [1]PF6 attains a distorted octahedral geometry with
GYY4137 coordinated trans to the biquinoline ligand. The
sterically demanding tpy and biq ligands perturb the Ru-ligand
bond angles, which range from 77.31° to 99.23°, significantly
from the 90° of an ideal octahedron. This steric strain
contributes to the efficient photosubstitution reactions of
these complexes by lowering the energy of the dissociative 3LF
state, allowing for it to be thermally populated from the
photogenerated 3MLCT state.52−55 The Ru−S1 distance (2.41
Å) is similar to that found in a related organometallic Ru(II)
complex bearing GYY4137 as a ligand (2.403 Å).56
S15, SI).44,45 The photosubstitution quantum yields (Φ626) at
room temperature for [1]Cl and [2]Cl were found to be similar
to those measured for related Ru(II) complexes (Table
1).42,44,45
The red light-induced photoreaction of [1]Cl was further
1
investigated by H and 31P{1H} NMR spectroscopy in 90/10
D2O/DMSO-d6. Upon irradiation, the characteristic doublet of
[1]Cl in the 1H NMR spectrum at 9.97 ppm, which is assigned
to the proton in the ortho position of the biq ligand directly
adjacent to the coordinated GYY4137, vanished while a new
quartet at 8.94 ppm, assigned to [Ru(tpy)(biq)(OH2)]2+, grew
in over time. Similarly, peaks originating from [1]Cl at 8.80 and
The photophysical properties of [1]Cl and [2]Cl in buffered
aqueous solution (3-morpholinopropanesulfonic acid; MOPS;
pH 7.4) were investigated (Table 1). The complexes exhibit
B
DOI: 10.1021/jacs.8b08695
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Journal of the American Chemical Society
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8.70 ppm were replaced with doublets corresponding to
[Ru(tpy)(biq)(OH2)]2+ at 8.63 and 8.59 ppm, (Figure S16,
SI). In the 31P{1H} NMR spectrum, the singlet arising from the
coordinated GYY4137 ligand of [1]Cl was replaced by a peak at
98.93 ppm, which is conclusively assigned to free GYY4137
(Figure S17, SI).
Ru(II) compounds can bind to DNA and induce cytotoxicity.54
As expected, GYY4137 is noncytotoxic (Figures S22 and S23,
SI).
Given the promising toxicity profile of [1]Cl and its
photoproducts, the H2S-releasing capabilities of this compound
were measured in living cells using the cell-trappable H2S-
responsive fluorescent probe, SF7-AM.58 Lung cancer (A549)
cells were loaded with SF7-AM and treated with the complexes,
followed by irradiation with red light for 30 min prior to
fluorescence microscopy imaging. Cells that were only treated
with [1]Cl or exposed to red light showed no significant increase
in fluorescence intensity of SF7-AM. In contrast, when both [1]
Cl and red light were administered to cells, a significant increase
in fluorescence intensity was observed, indicating that both
components are critical for the intracellular release of H2S
(Figure 4).
For these light-activated complexes to be valuable as stimuli-
responsive agents, they must not undergo thermal loss of the
phosphinodithioate ligand. As such, the thermal stabilities of
these complexes were assessed in aqueous buffer. We found that
[2]Cl is unstable in the dark, forming [Ru(tpy)(biq)(OH2)]2+
and GYYOMe via a thermal aquation reaction (Figure S18, SI).
In contrast, [1]Cl did not show appreciable changes in its
absorption spectrum after 5 days in solution (Figure 2),
indicating that this compound is sufficiently stable for use as a
light-activated H2S-releasing agent. Notably, the GYY4137
ligand did not hydrolyze while coordinated to the Ru(II) center.
Given the promising thermal stability and efficient photo-
substitution quantum yield of [1]Cl, we further investigated its
H2S-releasing properties. The formation of H2S in aqueous
buffer was evaluated using the fluorescent probe 1,5-dansyl azide
(DNS-az).57 The azide functional group of this probe is
selectively reduced by H2S to elicit a linear, fluorescent turn-on
response (Figure S19, SI). Using this probe, we observed
negligible release of H2S from [1]Cl in the dark. By contrast,
upon irradiation with 626 nm light, a steady time-dependent
increase in H2S concentration was detected, comparable to that
of free GYY4137 (Figure 3). By coordinating GYY4137 to the
ruthenium photocage in complex [1]Cl, we gain temporal
control over the otherwise continuous release of H2S from
GYY4137.
Figure 4. Images of H2S release from [1]Cl. A549 cells were incubated
with 5 μM SF7-AM for 30 min, washed and then (A) exposed to red
light for 30 min; (B) treated with 60 μM [1]Cl and exposed to red light
for 30 min; (C) incubated for 30 min in the dark; (D) treated with 60
μM [1]Cl for 30 min in the dark. (E) Mean pixel intensity of
fluorescence images for A549 cells in the experiments described in
panels A−D. Error bars are SE of n = 5 or 6 wells from two biological
replicates. Statistical significance was determined using a two-tailed
student’s t test. **p < 0.005.
Because H2S can protect against I/R injury,59−65 we
investigated the ability of [1]Cl to give rise to cytoprotective
effects selectively upon red light irradiation in H9c2 cells
subjected to hypoxia/reoxygenation injury. Cells were in-
cubated in a pH 6.4 ischemia mimetic buffer (see SI for details)
in hypoxic conditions, followed by treatment with [1]Cl or
GYY4137 in the dark or under red light irradiation for 30 min
prior to reoxygenation. Control cells were incubated in
normoxic conditions for the duration of the experiment.
The viability of untreated cells, measured by the colorimetric
MTT assay,66 subjected to this I/R injury model was decreased
by approximately 80%, indicating that this model accurately
captures the cytotoxic effects of this condition. When cells were
treated with [1]Cl in the dark, there was no significant change in
cell viability compared to untreated cells. In the presence of red
light, however, cells treated with [1]Cl show a 75% increase in
viability relative to the untreated control cells. In comparison,
the extent of the cytoprotective effect of GYY4137 does not
change appreciably when cells are treated either in the light or
dark (Figure 5). These results demonstrate that [1]Cl can
effectively cage GYY4137 and prevent H2S release until selective
red light activation to prevent cell death in a model of I/R injury.
In summary, we have developed a novel red light-activated
H2S-donating complex. Compared to other light-activated H2S-
Figure 3. H2S release from [1]Cl (200 μM) with red light irradiation
(red circles), [1]Cl (200 μM) in the dark (black squares), and
GYY4137 (200 μM) (purple triangles) measured by DNS-az
fluorescence. Errors bars are the standard error (SE) of three
independent trials.
For [1]Cl to be useful as a H2S-releasing biological tool or
therapeutic agent, it should not give rise to toxic effects. With
short (4 h) incubation times, conditions that are favorable for
H2S-releasing applications, [1]Cl is essentially nontoxic to
healthy cells (Table S3, Figures S20 and S21, SI). At longer
times, [1]Cl does show mild toxicity (Table S4, Figures S22 and
S23, SI). The aquated photoproduct [Ru(tpy)(biq)(OH2)]2+ is
effectively nontoxic, as indicated by its IC50 value of 97 4 μM,
consistent with values that have previously been measured for
this compound. These results are significant because related
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DOI: 10.1021/jacs.8b08695
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releasing systems, this compound is the first small molecule that
is capable of releasing H2S upon irradiation with red light
without the need for a secondary nanoparticle system. We have
shown that [1]Cl is stable in the dark and can be activated to
release H2S in biological conditions. In addition, [1]Cl is
capable of preventing cell death in a model of I/R injury. This
work demonstrates the relatively understudied cytoprotective
properties of transition metal compounds, and suggests a
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treatment and prevention of serious medical conditions.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.8b08695.
■
S
Complex characterization data, cell viability curves,
crystal data tables, UV−vis spectra, NMR data of
irradiation (PDF)
Data for[1]PF6 (CIF)
AUTHOR INFORMATION
Corresponding Author
*jjw275@cornell.edu
■
ORCID
Joshua J. Woods: 0000-0002-6213-4093
Justin J. Wilson: 0000-0002-4086-7982
Notes
(27) Steiger, A. K.; Marcatti, M.; Szabo, C.; Szczesny, B.; Pluth, M. D.
The authors declare no competing financial interest.
ACS Chem. Biol. 2017, 12, 2117−2123.
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Lett. 2017, 19, 62−65.
ACKNOWLEDGMENTS
■
(29) Zhao, Y.; Bhushan, S.; Yang, C.; Otsuka, H.; Stein, J. D.; Pacheco,
A.; Peng, B.; Devarie-Baez, N. O.; Aguilar, H. C.; Lefer, D. J.; Xian, M.
ACS Chem. Biol. 2013, 8, 1283−1290.
This work was supported by Cornell University and the National
Science Foundation (NSF-GRFP for J.J.W.; award no. DGE-
1650441). This work made use of the NMR facility at Cornell
University which is funded, in part, by the NSF (award no. CHE-
1531632). This work was additionally supported by the NIH
(award no. NIGMS R15GM114792 to A.R.L.).
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