Nitric oxide release from a photoactive water-soluble ruthenium nitrosyl. Biological?effects

Article abstract of DOI:10.1080/00958972.2018.1469129

This research presents the synthesis and characterization of the photochemical nitric oxide (NO) precursor Ru(salenCO₂H)(NO)Cl (1, salenCO₂H?=?N,N’-ethylenebis(3,3’-bis-carboxylsalicylideneiminato). This water-soluble ruthenium nitro

Full text of DOI:10.1080/00958972.2018.1469129

Journal of Coordination Chemistry
ISSN: 0095-8972 (Print) 1029-0389 (Online) Journal homepage: http://www.tandfonline.com/loi/gcoo20
NO Release from a photoactive water-soluble
ruthenium nitrosyl. Biological effects
Meredith A. Crisalli, Lilian P. Franco, Bruno R. Silva, Alda K. M. Holanda,
Lusiane M. Bendhack, Roberto S. Da Silva & Peter C. Ford
To cite this article: Meredith A. Crisalli, Lilian P. Franco, Bruno R. Silva, Alda K. M. Holanda,
Lusiane M. Bendhack, Roberto S. Da Silva & Peter C. Ford (2018): NO Release from a photoactive
water-soluble ruthenium nitrosyl. Biological effects, Journal of Coordination Chemistry, DOI:
10.1080/00958972.2018.1469129
To link to this article: https://doi.org/10.1080/00958972.2018.1469129
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Publisher: Taylor & Francis
Journal: Journal of Coordination Chemistry
DOI: http://doi.org/10.1080/00958972.2018.1469129
NO Release from a photoactive water-soluble ruthenium nitrosyl.
Biological effects#
MEREDITH A. CRISALLIa,##, LILIAN P. FRANCOb, BRUNO R. SILVAb, ALDA K. M. HOLANDAa,c,
LUSIANE M. BENDHACKb, ROBERTO S. DA SILVAb,* and PETER C. FORDa,*
aDepartment of Chemistry and Biochemistry, University of California, Santa Barbara,
Santa Barbara, CA 93106 USA
bFaculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av. do Café s/n,
CEP 14040-903, Ribeirão Preto, SP, Brazil
cDepartamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará Cx. Postal 12200,
Campus do Pici s/n, CEP 60440-900, Fortaleza - CE, Brazil
This research presents the synthesis and characterization of the photochemical nitric oxide (NO)
precursor Ru(salenCO2H)(NO)Cl (1, salenCO2H = N,N'-ethylenebis(3,3’-bis-
carboxylsalicylideneiminato). This water-soluble ruthenium nitrosyl releases NO upon
photolysis with a quantum yield that is pH dependent owing to the nitrosyl to nitrite conversion
of that axial ligand at higher pH. Also described are the water, oxygen, and thermal stability of 1
and the cytotoxicity and the vascular relaxivity properties of 1 in the dark and under photolysis.
Keywords: Ruthenium nitrosyl; Photochemical release; Nitric oxide; Myography; Cell viability
# This article has been submitted to honor the distinguished career of Prof. Dan Meyerstein.
## Taken in part from the Ph.D. Dissertation of MAC, University of California, Santa Barbara, 2015
* Corresponding authors. Email: silva@usp.br (R.S. da Silva); ford@chem.ucsb.edu (P.C. Ford)
1

1. Introduction
The photochemical "uncaging" of biologically active molecular species has drawn considerable
interest [1], since this methodology provides the spatially specific opportunity to release defined
concentrations of a substance such as nitric oxide (nitrogen monoxide) at a biological target. This
specificity may minimize systemic side-effects while controlling the timing of such an event
[2-6]. In this context, we [7-14] and others [15-27] have studied the photochemistry of ruthenium
nitrosyl compounds owing to their thermal stabilities and their photolabilities toward NO release.
Endogenously produced NO demonstrates key bioregulator roles in mammalian physiology
(including vasodilation) [28] as well as serving as a cyto-toxin at the higher concentrations
generated during immune response to pathogens [29-31]. Moreover, NO is a sensitizer of
γ-radiation damage to cells, a feature that could prove valuable in the radiotherapy of hypoxic
tumors [32-34]. Application of NO in the latter task is especially attractive given that the
concomitant vasodilation should also increase tissue oxygenation at the targeted site and that O2
is a radiation sensitizer as well. However, due to NO’s promiscuous bioactivity, controlling its
dosage will be crucial in such therapeutic applications [35], and this provides a major incentive
for developing photo-activated NO releasing moieties (photoNORMs) [36].
In this context, we have prepared a new photoNORM Ru(salenCO2H)(NO)Cl (1)
(salenCO2H = N,N'-ethylenebis(3,3’-bis-carboxyl-salicylidene-iminato) that is water soluble, is
thermally stable in aerated aqueous media, and is photolabile to NO release. Notably, water
solubility adds the challenge of pH dependence to the photoreactivity of 1 owing to the
equilibrium between the nitrosyl and N-nitrito forms of this ligand. Although this reversible
reaction for ruthenium nitrosyls has long been known [37-41], the present study is to our
knowledge, the first to characterize the photoactivated release of NO from such a pH dependent
photoNORM system. We also report the activity of 1 in cell toxicity and vascular reactivity
studies showing that 1 has moderate cytotoxicity before and after irradiation in cells as well as
being able to trigger vasodilation in mammalian aortic rings only in the presence of light with no
such effects observed in the dark.
2

2. Results and discussion
The carboxylic acid functionalized ruthenium salen nitrosyl complex Ru(salenCO2H)(NO)Cl (1)
was prepared according to the reactions outlined in scheme 1. Details are presented in the
Experimental section.
N
OH
OH
O
O
(i)
(ii)
OH
HO
N
N
HO
OH
O
O
N
N
N
O
OH
OH
2
3
NO
O
O
(iii)
O
O
HO
OH
Ru
N
N
Cl
1
Scheme 1. Synthesis of Ru(salenCO2H)(NO)Cl (1): (i) trifluoroacetic acid, under argon, 4 h
reflux; (ii) 0.5 equivalents of ethylenediamine in ethanol, 1 h reflux; (iii) 2.3 equiv. NaH, 1
equiv. Ru(NO)Cl3·H2O under argon in ethanol 4 h reflux.
The UV-visible spectrum of 1 in aqueous solution is quite sensitive to the pH (figure 1).
These reversible spectral changes can be attributed to two types of reactions,
deprotonation/protonation of the two ligand carboxylic acid groups (Eq. 1) and the acid/base
dependent nitrosyl ↔ nitrite conversion described above (Eq. 2). Ruthenium-based nitrosyl to
nitrite conversions have been studied by a number of researchers [37-41], who demonstrated the
reaction of the nitrosyl electrophile (formally represented as RuII(NO+) in such complexes) with
hydroxide.
(1)
(2)
[1-NO]2–
[1-NO2]4–
3

The pH dependences of these two transformations in 1 were determined by systematically
recording the solution spectrum at 100 separate pH values ranging from 0.5 to 11.5. The
carboxylic acid/carboxylate equilibrium(a) (Eq. 1) was(were) evidenced by spectral changes at
lower pH values. This was studied quantitatively by plotting the ratio of the absorbance at 247
nm to that at 428 nm (Abs277/Abs428) at pH values from 0.5 to 6. A single inflection in this curve
is evident between pH 2.9 and pH 4.3 indicative of a pKa value of ~3.65 (figure 2-top). Since
only one such inflection was observed, a pKa for a second carboxylic acid is not obvious. Since it
is very unlikely that this would occur below 0.5, the more likely explanation is that the first and
second carboxylate pKa values are very close, and the resulting spectral changes for dissociation
of the second carboxylic acid occur in tandem at approximately the same pH as those seen for
the first carboxylic acid as suggested by Eq. 1. Given that the photochemical and biological
experiments reported here were all carried out at pH values significantly above 4, we can assume
that under each set of conditions the carboxylates were fully dissociated.
Figure 2-bottom is a plot of the ratio of the absorbance at 371 nm to that at 323 nm
(Abs371/Abs323) vs. the solution pH over the range 3.5 to 11.5. This plot displays a clear
inflection at pH 7.65, a value that is consistent with the pKa of the nitrosyl/nitrite conversion (Eq.
–
2) based on earlier observations with analogous ruthenium nitrosyls. The NO+ to NO2
conversion ([1-NO]2– to [1-NO2]4–) was also apparent in the aqueous solution phase IR spectrum
which showed that the nitrosyl νNO peak at 1865 cm-1 disappears when the solid is dissolved in
alkaline solution but reappears when the solution is acidified.
All the pH dependent changes in the UV-visible spectra are reversible, suggesting that the
compound does not decompose readily at either low or high pH. This reversibility is consistent
with the equilibria shown in Eqs. 1 and 2.
2.1. Photochemical studies
Photolysis of buffered aqueous solutions of 1 at irr = 365 nm led to NO release as detected using
a Sievers Nitric Oxide Analyzer (NOA that quantitatively measures NO with high sensitivity.
Figure 3 shows a plot of the NO detected (in moles) versus the light absorbed (in Einsteins) by
the reactant. This plot is linear with a slope equal to NO. These quantum yields were measured
at different pH values in order to assess the effects of the pH-dependent equilibria discussed
above. The lower pH results are of particular interest, since the pH of cancer cells falls below 6.8
4

under hypoxic respiration [42]. NO photolabilization measured in this manner gave the modest
NO values (2.6±0.3) × 10–3, (4.8±0.3) × 10–3 and (5.2±0.2) × 10–3 for the respective pH values

7.4, 6.0 and 4.5. In analogy to earlier studies on ruthenium salen nitrosyl complexes [8, 10], we
interpret the principal photochemical reaction in terms of NO labilization from the nitrosyl form
[1-NO]2– (Eq. 3). Notably, the increased NO values at lower pH correspond to the higher
concentrations of the nitrosyl form [1-NO]2– relative to the N-nitrito analog [1-NO2]4– as
indicated by Eq. 2. Consistent with this view, it is notable that NO is markedly lower at higher
pH (0.9±0.2) × 10–3 and (0.5±0.1) × 10–3 at pH 9.0 and pH 11.0, respectively. Thus, it appears
that NO photogeneration from the N-nitrito complex is very much less efficient than from the
nitrosyl analog, as expected.
(3)
The ΦNO values for 1 at acidic pH are consistent with that (5×10–3) reported previously
for the aquo complex [Ru(salen)(H2O)(NO)]Cl (4) in aqueous solution when irradiated at 365
nm [10b]. However, both are surprisingly less photolabile than is the simpler, but water
insoluble, salen complex Ru(salen)(NO)Cl (5, salen = N,N’-ethylenebis(salicylideneiminato))
when irradiated in acetonitrile at 365 nm (ΦNO = 0.13 ± 0.01) [8b]. We do not have a ready
explanation for the >20 fold lower quantum yields for NO release from 1 and 4 in buffered
aqueous media than from 5 in acetonitrile, unless the solvent H2O somehow accelerates
nonradiative deactivation of the responsible excited state(s). Lehnert and coworkers [27] noted a
similar decrease in NO photolability of several other ruthenium nitrosyl complexes in aqueous
solution and pointed out that such decreases might affect the biological efficacy. The quantum
yield for NO release for photolysis carried out at 470 nm with a blue LED is even smaller (NO
=
0.2×10-3 in pH 7.4 buffered solution, SI figure S1).
2.2. Cellular viability
The cellular toxicity of 1 was studied using the murine melanoma B16F10 cell line. The cells
were incubated with concentrations of 1 ranging from 10 µM to 200 µM in Roswell Park
5

Memorial Institute 1640 medium (pH 7.4) and incubated for 4 h. The plates were then irradiated
at 470 nm with a blue LED for 4 min or kept in the dark as a control. All plates were then
incubated for either 24 or 48 h in the dark. The MTT assay ([40], see Experimental) was
performed to evaluate cell viability using wells not containing 1 as the controls (figure 4). The
cytotoxicity of 1 toward this cell line (B16F10) was moderate under the experimental conditions.
For example, there is no statistical difference between the control and the cells incubated with a
concentration of 10 µM over 24 h as determined by Tukey’s post test with cell viability at 94%
and 97% for dark and irradiated samples. However, at much higher concentrations (100 to 200
µM), the cell viability after 24 and 48 h did decrease to the 50-60 % level. For these higher
concentration experiments, there were modest, but statistically significant, increases in cell
viability for those cultures that had been exposed to light (SI figure S2). For example, cells
treated 200 µM 1 displayed 51% survival relative to the control after 48 h, while the analogous
samples that had been pre-irradiated with the 470 nm light had a 59% survival rate.
2.3. Vascular reactivity
Myography experiments were used to test the effect of Ru(salenCO2H)(NO)Cl on the
contraction/dilation of rat aortic rings from which the endothelium layer had been removed to
eliminate natural sources of NO generation. These experiments were carried out in pH 7.4 Krebs
solution over a large range in the concentration of 1 by the periodic addition of stock solutions of
this compound. The initial [1] was 10-10 M and the final concentration was 1×10–4 M. When the
system was kept completely in the dark, there was no effect on vascular relaxation over this
concentration range (figure 5). In contrast, under ambient room light, some vascular relaxation
was noted at [1] = 10-6 M, while full relaxation was verified when [1] equaled 10-5 M. However,
when a 10-5 M solution of 1 was added directly to fresh, fully contracted aortic rings under
ambient light (SI figure S3), full relaxation was slow, requiring ~50 min, owing to the
correspondingly slow photoreaction under these conditions. In the absence of light, very little
relaxation was observed, again consistent with the vasodilation process generally being triggered
by the activation of the very sensitive enzyme guanylyl cyclase [31]. The areas under these
curves were used to calculate the relative relaxation induced by 1 over 60 min in the dark and
under ambient light (SI figure S3). Notably, for the latter experiment, there was little significant
difference in the responses seen for thoracic aortas with their endothelium intact vs. those that
6

have been denuded. Thus, the vascular dilation observed under ambient light can be attributed to
the NO released by photolabilization from the ruthenium nitrosyl and not by residual NO from
endogenous production in the system.
2.4. Chemical and electrochemical reduction
Ruthenium nitrosyl complexes have also been shown to release NO upon one electron reduction
(Eq. 4) [44]. This type of release was tested with Ru(salenCO2H)(NO)Cl electrochemically, and
it was found that in order to induce the production of NO, 1 required a charging potential at –0.5
V vs. Ag/AgCl. Differential pulse voltammograms of 1 in aqueous solution shows the peak at –
0.3 V vs. Ag/AgCl attributed to {Ru-NO}2+/3+ oxidation after such charging. The initial reduction
with similar systems has been identified as centered on the nitrosyl ligand, which can be
represented as follows: RuII(NO+) + e–  RuII(NO•) [44]. Pre-treatment at –0.5 V causes
decrease in the peak intensity at –0.3 V (figure 6), consistent with labilization of the NO ligand
upon reduction of 1 (Eq. 4). A second peak at ~ 0.1 V vs. Ag/AgCl appears after the reduction
process, which was attributed to the oxidation of the aquo species Ru(salenCO2H)(H2O)Cl, [1-
H2O]3–, which is formed upon NO labilization from reduced 1. Thermal release of NO from
reduced 1 was qualitatively confirmed by using a NO specific electrode (SI figure S4). The time
dependence of the scans in figure 6 as well as for the formation of free NO suggest that thermal
release of NO from the reduced 1 (Eq. 4) has a half-life of several hundred seconds.
(4)
The addition of ascorbate (0.14 M) to a solution of 1 (28 μM) in pH 7.4 phosphate buffer
also triggers NO release as demonstrated using the NO specific electrode system (SI figure S4).
3. Summary
This study has described a photo-activated NO releasing moiety Ru(salenCO2H)(NO)Cl (1) that
is stable in aerobic aqueous solutions while displaying pH dependent quantum yields for NO
generation in vitro. This complex was shown to stimulate vasodilation in mammalian aortic rings
7

specifically when exposed to ambient light. The pH dependent quantum yields can be attributed
to the nitrosyl / N-nitrito equilibrium that results in enhanced NO release at lower pH. Such an
effect suggests a strategy for targeting NO release in the relatively acidic environment
characteristic of hypoxic tumors [45, 46]. Neither the photoNORM precursor nor the small
amount of NO released is cytotoxic, but the photo-induced NO release, even with a very modest
quantum yield, would be sufficient to trigger soluble guanylyl cyclase mediated vasodilation
[47], thereby increasing circulation to and oxygenation of the targeted tissues. If this process
were coupled to radiotherapy, the increased oxygenation would enhance the sensitivity of the
targeted tissues to radiation-induced cell death. Similarly, such targeted NO release leading to
enhanced circulation could synergistically enhance the efficacy of chemotherapy.
Nonetheless, a realistic evaluation of the present system has to take into consideration
that the short wavelength necessary to trigger NO release from 1 would have poor penetration
through living tissue. For this reason, strategies that can utilize tissue penetrating near infrared
(NIR) light such the use of antennas such as upconverting nanoparticles that can be activated by
multiphoton NIR excitation need to be employed. Notably, the pendant carboxylate groups of 1
besides providing aqueous solubility, also provide ample opportunity for further conjugation
with groups like cell-targeting peptide [48] or with antennas such as nanomaterials with desirable
photophysical properties [13, 49]. These studies are ongoing.
4. Experimental
4.1. Materials
Trichloridonitrosylruthenium(III) monohydrate, Ru(NO)Cl3·H2O was purchased from Strem
Chemicals Inc. Other synthetic materials including trifluoroacetic acid, 4-hydroxybenzoic,
hexamethylenetetramine, 95% sodium hydride and laboratory solvents were purchased from
Sigma-Aldrich. Cell lines, media, and MTT assay reagents for cell toxicity studies were provided
by the Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo. All
buffers used for quantum yield measurements were prepared in nanopure water at 50 mM
concentrations. The pH was adjusted to exact values using dilute NaOH or HCl as required.
Phosphate buffers were prepared for pH values 11.0, 7.4, 6.0, and 4.5 while
tris(hydroxymethyl)aminomethane (Tris) was used to prepare the pH 9.0 buffer.
8

4.2. Synthesis of Ru(salen-CO2H)(NO)Cl (1)
The salen-CO2H ligand (3) was prepared according to the reaction sequence outlined in scheme 1
from salicylaldehyde-4-carboxylic acid (2), which was synthesized according to published
procedures [50] (see SI). The nitrosyl complex chloridonitrosyl(N,N’-ethylenebis(3,3’-bis-
carboxylsalicylidene-iminato)ruthenium(III) (1) was prepared from 3 as follows. A mixture of 3
(0.50 g, 1.41 mmoles), sodium hydride (0.107 g, 4.45 mmoles), and
trichloridonitrosylruthenium(III) monohydrate, Ru(NO)Cl3·H2O (0.317 g, 1.41 mmoles), was
added to a three neck 300 mL round bottom flask and placed under an argon atmosphere.
Approximately 150 mL of deaerated ethanol was then transferred via cannula into the reaction
flask. The resulting solution was refluxed under argon in the dark for 4 h, after which it was
allowed to cool to room temperature, and the ethanol was removed via rotary evaporation. The
sodium chloride byproduct was removed from the desired product by washing the solid with
water adjusted to pH 5 with dilute HCl. The remaining solid was dried under vacuum for several
hours (scheme 1, step iii) (89% yield). 1H NMR (DMSO-d6): δ 8.69 ppm (1H, s) aldimine
protons, 8.02 (1H, s), 7.85 (1H, d), 6.83 (1H, d) phenyl protons, 3.94 (2H, s) ethylene bridge.
Analysis: Theoretical for [Ru(salenCO2H)(NO)Cl]·2H2O (C18H16N3O7ClRu·2H2O): C, 38.68%;
H, 3.61%; N, 7.52%. Found: C, 38.3%; H, 3.43%, N 7.59%. FTIR (KBr) νNO = 1865 cm-1. UV-
vis (pH 7.4 in 50 mM phosphate buffer): max 248 nm ( = 3.8×104 M-1 cm-1), 359 ( = 6.6×103
M-1 cm-1).
Once the product was washed with the pH 5 solution, solubility for aqueous solution
experiments was achieved by dissolving the compound into a solution above pH 9. These stock
solutions were then diluted into buffered media for spectroscopic studies or for investigation
with murine tissues or cells.
4.3. Quantum yield measurements
NO release was measured using a GE model NOA 280i Sievers Nitric Oxide Analyzer (NOA)
calibrated per the manufacturer's specifications. This device quantitatively measures the NO
present in a carrier gas, which is either helium or medical grade air, that is entrained through the
photolysis solution [51]. The carrier gas was first passed through a glass chamber containing
only solvent prior to entering the reaction cell in order to prevent evaporative solvent loss and to
maintain a constant volume in the reaction cell throughout the experiment. Quantum yield
9

measurements at the pH values at 4.5, 6.0, 7.4, 9.0, and 11.0 were conducted individually using a
stock solution of 1. The cuvette was filled with 3 mL of pH-adjusted solutions of 1 and entrained
with carrier gas for 5 min in order to allow the NOA establish a stable baseline. The photolysis
was conducted at irr 365 nm with the light from a high-pressure mercury lamp passed through a
narrow band pass interference filter or at irr 470 nm with a blue LED from Luxeon. The
intensity of the excitation at 365 nm was determined using ferrioxolate actinometry [52], and the
intensity of the LED light was measured with a Newport power meter. Individual samples were
irradiated for varied time intervals controlled by a Uniblitz shutter and the photochemically
released NO was recorded using the NOA software “Liquid”. The plotted slope is NO (see
figure 3).
4.4. pKa Measurements
The pH dependent changes in UV-visible spectrum of 1 were recorded in order to examine the acid/base
dependent equilibria. In order to determine these quantitatively, 1 was dissolved in slightly basic water
and diluted to a concentration of 0.15 mM in 50 mM KCl solution. The pH was adjusted by adding small
increments of dilute hydrochloric acid and determined with a pH meter. After the pH stabilized, the UV-
visible spectrum was recorded. This was done for ~100 different pH values from 0.5 to 11.
4.5. Cell viability studies
The cytotoxicity of 1 was evaluated against B16F10 murine melanoma cells using the
colorimetric method with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT
assay) [43]. The cells were cultured in Roswell Park Memorial Institute 1640 medium (RPMI)
enriched with 2% fetal bovine serum, seeded into 96-well plates (2×105 cells/well) and incubated
according to published procedures [53]. Stock solutions of 1 were prepared in concentrations of
20 mM, 10 mM, 2 mM, and 1 mM, and 2 µL aliquots of these solutions were added to each well
to obtain a total volume of 200 µL of media. The final concentrations of 1 in the wells were 200
µM, 100 µM, 20 µM, 10 µM, and 0 µM (control). The cells were incubated with 1 in the dark for
4 h in the RPMI medium (pH 7.2). Phenol red indicator was not used in the media during the
photolysis to avoid light being absorbed by this indicator. Half of the cell plates were irradiated
with 470 nm light for 4 min with a modified cell plate LED, and all plates were then incubated
10

for either 24 or 48 h. After the incubation, the media were removed and 200 µL MTT solution
(0.5 mg/mL RPMI medium) was added to each well and incubated for 3 h. MTT is converted
into crystals by the viable cells. To solubilize the product, the medium was removed and 200 µL
of DMSO was added into the wells. (MTT solution; 12 mM thiazolyl blue tetrazolium bromide
in phosphate buffer was diluted in media to a final concentration of 1.2 mM.) The absorbance
was recorded by a Thermo Plate leitora de microplaca TP-reader plate reader at 570 nm. The
reported values are an average of triplicate samples in three different experiments. The data was
normalized to the control wells, not containing 1. Data was compiled and processed using Prism
5.0 software.
4.6. Vascular dilation studies
All the procedures were performed in accordance with the standards and policies of Animal Care and Use
Committee of the University of São Paulo. Male rats (180-200 g) were sacrificed by decapitation under
anesthesia. The thoracic aorta was quickly removed and cut into rings (4-5 mm length). The endothelium
was mechanically removed from one of the two rings by gently rolling the lumen of the vessel on a thin
wire. Aortic rings with and without endothelium cells were studied concurrently. The aortic rings were
placed between two stainless-steel stirrups and connected to an isometric force transducer (Letica
Scientific Instruments, Barcelona–Spain) to measure tension in the vessels. The rings were placed in an
organ chamber containing Krebs solution with the following composition (mM): NaCl (130.0), KCl (4.7),
KH2PO4 (1.2), MgSO4 (1.2), NaHCO3 (14.9), glucose (5.5) and CaCl2 (1.6). The solution was maintained at
pH 7.4 and bubbled with a gas mixture of 95% O2 and 5% CO2 at 37 °C. The rings were initially stretched
to a basal tension of 1.5 g and allowed to equilibrate for 60 min. The presence of endothelium was
evaluated by the degree of relaxation induced by acetylcholine (1 µM) in the presence of contractile
tone induced by phenylephrine (PE, 0.1 µM). Endothelium-intact arteries were discarded if relaxation
with acetylcholine was not greater than 80% while endothelium-denuded arteries were discarded if
there was degree of relaxation for acetylcholine higher than 10%. The rings were then washed and pre-
contracted with 10 µL of PE (0.1 µM). After stabilized contraction, two tests for vascular relaxation were
separately conducted using 1: the concentration-effect curve and the time course with the necessary
concentration to induce maximum vascular relaxation. For the concentration-effect curve, stock
solutions from 0.1 µM - 5 mM were prepared and cumulatively added in sequence to the Krebs buffered
organ chambers. The final concentration ranged from 10-10 M to 10-4 M (figure 4). The time course for 1
11

induced relaxation was monitored over the course of an hour following the single administration of 20
µL of 5 mM 1 for a final concentration of 10-5 M (figure 5). Both experiments were conducted in ambient
light or dark. The comparisons between groups were assessed by student t-test. The level of statistical
significance was defined as P<0.05.
4.7. Voltammetric studies
The electrochemical study was performed with an AUTOLAB PGSTAT 30 model potentiostat galvanostat
consisting of a conventional three electrode cell with a platinum wire auxiliary electrode, an Ag/AgCl
reference electrode, and a glassy carbon working electrode. An aqueous 0.1 M KCl solution was used as
the supporting electrolyte. The solution was charged at -0.5 V for a period of 0 and 300 s prior to
scanning from -0.5 V - 0.5 V. Additionally, negative scan was charged at 0.3 then run over from 0.3 - -
0.5 V.
Author contributions
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript.
Supporting information
Three pages with 4 additional figures and the synthesis of the ligand salenCO2H.
Acknowledgements
This research was supported by the US National Science Foundation (CHE-1058794 and
CHE-1405062) for work completed in the US and by the Brazilian agencies Conselho Nacional
de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de
Pessoal de Nível Superior (CAPES) and Fundação de Amparo à Pesquisa do Estado de São
Paulo (FAPESP) for work completed in Brazil. M.A.C. thanks the ConvEne-IGERT program at
12

UCSB (NSF-DGE 0801627) for a fellowship and for travel support to carry out experiments in
Brazil.
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Figure 1. The optical spectra of Ru(salenCO2H)(NO)Cl (1) recorded in 50 mM phosphate
solution at pH 1.0 (purple short dashes), pH 7.04 (black solid line) and at pH 11.07 (red long
dashes). At pH 7.4 the max appear at 229 nm ( = 1.13 × 104 M-1 cm-1), 248 nm (1.18 × 104 M-1
cm-1) and ~ 364 nm (2.8 × 103 M-1 cm-1).
16

Figure 2. Top: Relative absorbance changes for 1 over the pH range 0.5 to 6 plotted as the ratio
Abs247/Abs438, where Abs247 is the absorbance at 247 nm and Abs438 is the absorbance at 438 nm. These
are interpreted as indicating the pKa of the carboxylic acid functional group(s) of Ru(salenCO2H)(NO)Cl.
Bottom: Spectral titration over the pH range 3.5 to 11.5 of the absorbance ratio Abs371/Abs323, where
371 nm is max for the nitrite form and 323 nm is an isosbestic point for all forms of 1. This is interpreted
as reflecting the pH dependence of the N-nitrito/nitrosyl equilibrium for 1. Conditions for both
experiments: [1] = ~1 × 10–4 M in 50 mM KCl solution at 298 K.
17

Figure 3. NO release from Ru(salenCO2H)(NO)Cl (1) under 365 nm irradiation at pH 4.5 (blue
diamond), 6.0 (orange square), 7.4 (green triangle), and 11.0 (purple circle), phosphate buffer
solution (50 mM). The plot shows NO (in moles) produced as determined by the NOA verses the
light absorbed Ia*t, where Ia is the intensity of the light absorbed in Einsteins per s and t is time
in s). The quantum yield NO is the slope of the line and equals 5.3 × 10–3 (pH 4.5), 2.2 × 10–3
(pH 6.0), 2.6 × 10–3 (pH 7.4), and 0.5 × 10–3 (pH 11.0).
18

Figure 4. Cell viability plots in different concentrations of 1 (Ru(salenCO2H)(NO)Cl). Cell
viability as assessed by the MTT assay (see Experimental). The results show cell survival
relative to the control (absence of 1). The values are the mean ± SEM. * p < 0.05 in comparison
to other treatments and the control obtained by ANOVA analyses using Tukey’s pos test. The
plates under light were irradiated for 4 min at 470 nm (4 J/cm2) at the beginning of the
experiment.
19

Light
Dark
0
25
*
50
75
100
-11 -10
-9
-8
-7
-6
-5
-4
-3
Log [HOOC-Ru-NO] mol/L
Figure 5. Vascular relaxation induced by Ru(NO)(salenCO2H)Cl (1) in denuded rat aortic rings
in ambient light or in the dark. Data are mean ± SEM (n=6).
Figure 6. Differential pulse voltammetry scans with carbon glass electrode with Ag/AgCl
reference electrode of Ru(salenCO2H)(NO)Cl (1). Scans recorded after specified timed charging
with conditioning potential of -0.5 V and run between –0.5 and 0.5 V.
20

Graphical abstract
21