Trans-[Ru(NO)(NH3)P(O-)(OEt)2] 2+: A new and robust NO/HNO-donor in aqueous media

Article abstract of DOI:10.1016/j.ica.2014.05.010

This study describes the synthesis and reactivity in aqueous media of a new potential NO/HNO-donor: trans-[Ru(NO)(NH₃)₄P(O ^-)(OEt)₂](PF₆)₂. This compound exhibits a remarkable robustness over a wide range of pH relative to other similar ruthenium phosphorus nitrosyl complexes. At pH 3.0 and 25 °C, trans-[Ru(NO)(NH₃)₄P(O^-)(OEt) ₂](PF₆)₂ decays, yielding free diethyl phosphite with a half-life of 9 days (k = 8.9 × 10^-7 s ^-1). At pH 7.5 and 25 °C, this complex is competitively consumed by diethyl phosphite dissociation (k = 5.15 × 10^-5 s ^-1) and nucleophilic attack at the nitrosyl group (k = 7.25 × 10^-5 s^-1). Nevertheless, the trans-[Ru(NO)(NH ₃)₄P(O^-)(OEt)₂](PF₆) ₂ exhibits a half-life of 1.5 h (pH 7.5 and 25 °C) for delivering NO/HNO by electrochemical activation at potentials of -0.50 and -0.80 V versus SCE. The NO liberation from trans-[Ru(NO)(NH₃)₄P(O ^-)(OEt)₂]⁺ ion occurs with k_{- NO} = 0.24 ± 0.01 s^-1, and electrochemical data indicate that k_-HNO ? k_-NO.

Full text of DOI:10.1016/j.ica.2014.05.010

Accepted Manuscript
trans-[Ru(NO)(NH3)P(O-)(OEt)2]2+: A new and robust NO/HNO-donor in aque-
ous media
Daniela R. Truzzi, Douglas W. Franco
PII:
S0020-1693(14)00285-0
http://dx.doi.org/10.1016/j.ica.2014.05.010
ICA 15992
DOI:
Reference:
Inorganica Chimica Acta
To appear in:
Received Date:
Revised Date:
Accepted Date:
14 February 2014
21 April 2014
9 May 2014
Please cite this article as: D.R. Truzzi, D.W. Franco, trans-[Ru(NO)(NH3)P(O-)(OEt)2]2+: A new and robust NO/
HNO-donor in aqueous media, Inorganica Chimica Acta (2014), doi: http://dx.doi.org/10.1016/j.ica.2014.05.010
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trans-[Ru(NO)(NH3)P(O-)(OEt)2]2+: A new and robust NO/HNO-donor in aqueous
media
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Daniela R. Truzzia; Douglas W. Francoa*
a
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Departamento de Química e Física Molecular, Instituto de Química de São Carlos,
Universidade de São Paulo, Av. Trabalhador São-carlense, 400, Centro, São Carlos, São
Paulo, Brazil.
*Address inquiries to this author, email: douglas@iqsc.usp.br
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Keywords: Ruthenium complex / Nitric oxide / Nitroxyl / Phosphite
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Abstract
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This study describes the synthesis and reactivity in aqueous media of a new potential
NO/HNO-donor: trans-[Ru(NO)(NH3)4P(O-)(OEt)2](PF6)2. This compound exhibits a
remarkable robustness over a wide range of pH relative to other similar ruthenium phosphorus
nitrosyl complexes. At pH 3.0 and 25°C, trans-[Ru(NO)(NH3)4P(O-)(OEt)2](PF6)2 decays,
yielding free diethyl phosphite with a half-life of 9 days (k = 8.9 × 10-7 s-1). At pH 7.5 and
25°C, this complex is competitively consumed by diethyl phosphite dissociation (k = 5.15 ×
10-5 s-1) and nucleophilic attack at the nitrosyl group (k = 7.25 × 10-5 s-1). Nevertheless, the
trans-[Ru(NO)(NH3)4P(O-)(OEt)2](PF6)2 exhibits a half-life of 1.5 h (pH 7.5 and 25°C) for
delivering NO/HNO by electrochemical activation at potentials of -0.50 and
-0.80 V vs SCE. The NO liberation from trans-[Ru(NO)(NH3)4P(O-)(OEt)2]+ ion occurs with
k-NO = 0.24 0.01 s-1, and electrochemical data indicate that k-HNO >> k-NO
.
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1. Introduction
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Ruthenium nitrosyl complexes of the type trans-[Ru(NO)(NH3)4L]3+ are a promising
nitric oxide donor (NO-donor) platform [1, 2]. In vitro and in vivo tests have successfully
established their activity in the hippocampus [3], aorta rings [4-6] and against Chagas disease
[7-9], Leishmania major [10] and cancer cells [11]. Among the tested compounds are those in
which L = 4-pic, py, pz, imN, imC, nic, ina and P(OEt)3 [2, 6, 7, 9-12]. Their biological
activity is attributed to their NO-donor ability upon reduction, which can be modulated
through the judicious choice of L. Phosphorus(III) ligands, such as phosphites (P(OR)3) and
phosphines (P(R)3) are interesting due to their high trans effect and trans influence [12-15]
+
properties, which can tune the reduction potential (ENO /NO) and the specific rate constant for
NO liberation (k-NO) of the NO+ fragment in these nitrosyl complexes [4, 5, 14]. Furthermore,
due to the possibility of varying the nature of R, additional tuning of the phosphorus atom σ
and π donor/acceptor abilities and the complex solubility can be achieved.
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+
For example, trans-[Ru(NO)(NH3)4P(OEt)3](PF6)3 exhibits ENO /NO = -0.24 V vs SCE
and k-NO = 0.97 s-1 [14], characteristics that are relevant for situations in which fast nitric
oxide liberation is required. In addition, after NO-donation, the resulting complex ion
-
trans-[Ru(H2O)(NH3)4P(OEt)3]2+ is able to react with nitrite (NO2 ) and, through an acid-base
-
equilibrium, convert NO2 into NO+ ligand [16], renewing the NO source. This system would
be very interesting in hypoxic conditions, where oxygen-dependent NO synthases may be
inhibited, by providing an alternative pathway for the production of nitric oxide through
nitrite conversion [17]. It is interesting to recall that nitrite is present in plasma at an average
concentration of 114 11 nmol L-1 [18], and the reduction potential of the [RuNO]3+ fragment
is accessible to biological reductors [9, 10, 14].
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Despite the interesting characteristics of trans-[Ru(NO)(NH3)4P(OEt)3](PF6)3 as a
NO-donor, it is not stable for practical application in biological media (pH ≥ 5) due to
nucleophilic attack at the triethyl phosphite ligand and at the nitrosonium group [19],
decreasing the NO-donation efficiency of this compound.
Recently, phosphorous acid (the simplest phosphite) was chosen as a probe in which
nucleophilic attack on the phosphite molecule is avoided by the absence of carbon chains
[20]. In this case, the trans-[Ru(NO)(NH3)4P(O-)(OH)2]2+ undergoes isomerization promoted
by
the
nitrosyl
group,
yielding
trans-[Ru(NO)(NH3)4(O)P(OH)2]2+
and
trans-[Ru(NO)(NH3)4(O)P(H)(OH)2]3+ ions in aqueous media. This isomerization reaction
also would reduce the compound NO-donor efficiency [20].
Therefore, this work presents a synthesis and reactivity study in aqueous media of the
new trans-[Ru(NO)(NH3)4P(O-)(OEt)2](PF6)2 complex. Diethyl phosphite is an interesting
alternative to the phosphorus(III) ligand because, unlike triethyl phosphite and phosphorous
acid, it maintains both the dissociable proton (hydroxyl group) and the ethyl groups (electron
donors).
2. Experimental Section
2.1 Chemicals and reagents
All chemicals were of analytical grade (Sigma-Aldrich, Strem or Merck). Ruthenium
trichloride (RuCl3·3H2O) was the starting reagent for the synthesis of the ruthenium nitrosyl
complex described herein. The solvents were purified following literature procedures [21]. All
syntheses and manipulations were carried out under an argon atmosphere [22].
2.2 Synthesis of the complexes
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The trans-[Ru(NH3)5Cl](Cl)2 [23] and trans-[Ru(H2O)(NH3)5](PF6)2 [24] complexes
were synthetized according to literature procedures.
2.2.1 trans-[Ru(NO)(NH3)4P(O-)(OEt)2](PF6)2 synthesis
First, 3.4 × 10-4 mol of trans-[Ru(H2O)(NH3)5](PF6)2 (169 mg) and 3.9 × 10-4 mol of
diethyl phosphite (0.5 mL) were added into 15 mL of argon-degassed acetone. After 1 h, NO
was bubbled into the solution for 4 h. Next, the acetone was evaporated, and 100 mg of
NH4PF6 and 50 mL of ether/ethanol (10:1) were added. The light red solid obtained was
separated through filtration, dried and stored under vacuum and the absence of light.
Yield: 40%.
Theoretical elemental analysis for trans-[Ru(NO)(NH3)4P(O-)(OEt)2](PF6)2·1/2CH3CH2OH:
C 9.25, H 3.88 and N 11.09. Found: C 9.23, H 3.71 and N 11.05.
2.3 Instruments
UV-vis spectra were acquired using a Hitachi U-3501 instrument, 1.0-5.0 cm quartz
cells and a thermostatic bath at 25 0.1°C.
Infrared spectra were obtained using a Bomem-102 instrument using KBr pellets (32
scans) for solid analysis and a silicon window with a 0.05-mm Teflon spacer (8 scans) for
solutions at a resolution of 2 cm-1.
Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were performed
in a Princeton Applied Research 264A instrument. The electrochemical cell was composed of
a saturated calomel electrode (SCE), a platinum plate and a glassy carbon electrode as the
reference, auxiliary and working electrodes, respectively.
The specific rate constant for NO liberation (k-NO) was calculated using the Nicholson
and Shain electrochemical method [25] at pH 2.0, µ = 0.1 mol L-1 and 25
0.1°C.
Considering the NO dissociation from trans-[Ru(NO)(NH3)4L]n+ fragments as a charge
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transfer followed by an irreversible chemical reaction, the specific rate constant for the NO
liberation (k-NO) was calculated based in the Ipa/Ipc ratio with the scan rate variation [25]. The
slope of the plot of k.τ (obtained from the working curve [25]) versus τ gives the k-NO value.
NMR spectra were acquired in a Agilent 500/54 Premium Shielded instrument using a
5-mm probe and 3-(trimethylsilyl)-2,2',3,3'-tetradeuteropropionic acid (TMSP-D4) (δ1Η,13C
=
0 ppm) or NH4PF6 salt (δ31P = -144 ppm) as internal references and D2O as the solvent. All of
the solutions were degassed using argon, their pH and strength ionic were controlled. For the
solutions at pH 7.5, tris(hydroxymethyl)aminomethane (TRIS) buffer was used. Unless
otherwise noted, trifluoroacetic acid (HTFA) was employed for pH adjustment.
2.4 Kinetic measurements
The trans-[Ru(NO)(NH3)4P(O-)(OEt)2]2+ ions were analysed in aqueous medium at pH
3.0 and 7.5 using 31P NMR, UV-vis and IR. All experiments were carried out at 25°C. The
data were modelled as a first-order reaction, with k being estimated from ln(A-At) vs time.
2.5 Computational details
The Gaussian03 [26] package was used to perform the calculations. The Kohn–Sham
density functional theory (DFT) [27] with the Becke three-parameter hybrid exchange-
correlation functional (B3LYP) and the standard DGAUSS basis set (DGDZVP for Ru,
DGDZVP2 for P and DGTZVP for H, C, N and O) was used to carry out the molecular
geometry optimizations [28, 29]. Symmetry conditions were not imposed [30]. The IEPCM
model of solvation with water as the solvent was used [31-33]. The vibrational frequencies
were carried out using the same method and the basis set under the keyword freq and did not
exhibited imaginary frequencies indicating a true minimum. The orbital population was
obtained from pop = full keyword and the electronic spectra were obtained using the TD-DFT
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calculation as implemented in the Gaussian03 package. The NBO 3.0 programme
implemented in the Gaussian03 package [34, 35] was used for natural bond orbital
calculations. For all of the structures calculated herein, the orientation is as follows: the
(NO)–Ru–(P(OH)(OEt)2) vector is the z-axis, and the x and y-axis correspond to the H3N–
Ru–NH3 vectors.
3. Results and Discussion
3.1 Trans-[Ru(NO)(NH3)4P(O-)(OEt)2](PF6)2 characterization
Trans-[Ru(NO)(NH3)4P(O-)(OEt)2](PF6)2 was isolated as an EPR-silent orange solid.
The orbital composition for this complex showed that the HOMO is 94% localized in Ru (dxy)
and the LUMO+0,1 are composed of 23% Ru (dxz, dyz) and 74% NO (px, py; π*). The Ru-
N=O angle obtained from DFT calculations is 175°, which strongly suggests that the NO
ligand has nitrosonium character [2].
The [Ru(NO)]3+ fragment exhibits a Ru(II) metal center with Ru(III) character. Indeed,
the pKa for water and phosphorous acid molecules in trans-[Ru(NO)(H2O)(salen)]+ (pKa =
4.50) [36] and trans-[Ru(NO)(NH3)4(P(OH)3)]3+ (pKa = 0.74) [20] are slightly smaller than
those in the respective aqua-ruthenium(III) complexes: trans-[Ru(H2O)2(salen)]+
(pKa = 5.90) [36] and trans-[Ru(H2O)(NH3)4(P(OH)3)]3+ (pKa = 1.0) [37]. Thus, the pKa for
diethyl phosphite molecule in trans-[Ru(NO)(NH3)4P(OH)(OEt)2]3+ is estimated to be of the
same order of magnitude as that in trans-[Ru(H2O)(NH3)4P(OH)(OEt)2]3+
(pKa = 1.5) [37]. According to UV-vis experiments (Fig. 1S), a band enlargement with an
increase of absorbance at 270 and 360 nm and, a small blue shift of ∆ = 4 nm are observed
when the solution pH changes from 1.0 to 3.0. This could be an indicative of the diethyl
phosphite ligand deprotonation which would occurs at pH higher than 2.0, but it does not
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enable an accurate pKa calculation. Therefore, in this study, in aqueous medium at pH > 2.0,
the diethyl phosphite in trans-[Ru(NO)(NH3)4P(O-)(OEt)2]2+ will be depicted in its
deprotonated form because it will certainly be the dominant form present under these
conditions.
The electronic spectrum of trans-[Ru(NO)(NH3)4P(O-)(OEt)2]2+ in solution at pH 2.0,
µ = 0.1 mol L-1 (Fig. 2S), similarly to those observed for other compounds of the
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trans-[Ru(NO)(NH3)4L]Xn series [38, 39], exhibited bands at
λ
~
530 nm
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(ε ~ 20 L mol-1 cm-1), 310 nm (ε = 956 L mol-1 cm-1) and 242 nm (ε = 1509 L mol-1 cm-1).
According to TD-DFT calculations, the absorption at 530 nm is assigned to the metal-
ligand charge transfer (MLCT) HOMO(dxyRu) ꢀ LUMO+0,1(π*NO), while the 310 nm
band would be a mixture of ligand-ligand charge transfer (LLCT), (P(O-)(OEt)2)HOMO-1 ꢀ
LUMO+0,1(π*NO)
and
MLCT
HOMO-3,4(dxz,dyzRu)
ꢀ
LUMO(π*NO).
The most intense band at 242 nm is mainly composed by an LLCT
(P(O-)(OEt)2)HOMO-2,5 ꢀ LUMO+0,1(π*NO) and a d-d transition of the HOMO-
3,4(dxz,dyzRu) ꢀ LUMO+2,3(dz2dx2-y2Ru) type.
Although the diethyl phosphite ligand presents a dissociable proton, the infrared
spectra of the nitrosyl complex of diethyl phosphite in the solid state (KBr) and in aqueous
medium (hydrogen ion concentration of 2.0 mol L-1 to 10-8 mol L-1) exhibited just one νNO+
at 1887 cm-1. This is different from the observations for the protonated and deprotonated
forms of trans-[Ru(NO)(NH3)4P(OH)3]3+/trans-[Ru(NO)(NH3)4P(O-)(OH)2]2+ ions, which
exhibit two νNO+ (1892 and 1879 cm-1), one for each species [20]. The DFT
calculation
for
the
structures
trans-[Ru(NO)(NH3)4P(OH)(OEt)2]3+
and
trans-[Ru(NO)(NH3)4P(O-)(OEt)2]2+ yielded NO+ frequencies at 1893 and 1883 cm-1,
respectively. These values are similar to one another, suggesting the possibility of band
overlap in the experimental spectra. Indeed, the plot of both theoretical νNO+ presented a
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bandwidth at half-maximum (w1/2) of 22 cm-1, which is the same value observed
experimentally for νNO+ = 1887 cm-1 in the title complex (Fig. 3S).
The 31P NMR spectra for trans-[Ru(NO)(NH3)4P(O-)(OEt)2]2+ obtained in solution
from 2.0 mol L-1 HTFA until reaching pH 7.5 exhibited just one singlet at 68 ppm (Table 1).
This single chemical shift is different from that observed when phosphorous acid (P(OH)3) is
bonded to [Ru(NO)(NH3)4]3+. In the last case, a singlet at 68 ppm for the protonated form and
a singlet at 60 ppm for the deprotonated form can be observed [20]. However, for the diethyl
complex, the NBO charges from DFT calculations show that the charge on the
phosphorus atom does not change substantially after the deprotonation of
trans-[Ru(NO)(NH3)4P(OH)(OEt)2]3+ (1.150) to form trans-[Ru(NO)(NH3)4P(O-)(OEt)2]2+
1
(1.158), thus corroborating the experimental data. Table 1 shows the H, 13C and 31P NMR
data for trans-[Ru(NO)(NH3)4P(O-)(OEt)2]2+ in D2O.
Insert Table 1
The
cyclic
voltammograms
for
solutions
containing
trans-[Ru(NO)(NH3)4P(O-)(OEt)2]2+ are depicted in Fig. 1a. The nitrosonium ligand is
reduced at Ecp = -0.50 V vs SCE (reaction 1), and the nitric oxide ligand is then reduced at Ecp
= -0.80 V vs SCE (reaction 2). Considering that the pKa for HNO molecule in
trans-[Ru(HNO)(NH3)4P(OEt)3]2+ estimated from DFT calculations is 9.9 [40] and
P(O-)(OEt)2 exhibits
a
π-acidity less than that of P(OEt)3 [12], in
trans-[Ru(HNO)(NH3)4P(O-)(OEt)2]2+, the HNO molecule probably exhibits pKa ≥ 10. Thus,
in solutions with pH < 10, the second reduction process (reaction 2) would yield
trans-[Ru(HNO)(NH3)4P(O-)(OEt)2]2+ instead of trans-[Ru(NO-)(NH3)4P(O-)(OEt)2]+. The
NO/NO+ and HNO/NO oxidation waves are only partially observed in Fig. 1a at Eap = -0.42 V
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and Eap = -0.63 V vs SCE due to the high trans effect of the phosphite ligand, which leads to a
fast NO/HNO dissociation. The reversible couple observed at 0.29 V vs SCE is attributed to
the RuIII/RuII couple in the aqua complex (reaction 3).
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Insert Figure 1
Applying the approach of Nicholson and Shain [25], the k-NO value for
trans-[Ru(NO)(NH3)4P(O-)(OEt)2]+ ion was calculated as 0.24 0.01 s-1 at pH 2.0 and 25
0.1°C (Fig. 4S). This value is four times smaller than the value measured for
trans-[Ru(NO)(NH3)4P(OEt)3]2+ (k-NO = 0.97 s-1 [14]). The k-HNO (the specific rate constant
for nitroxyl liberation) for this complex was not calculated using the Nicholson and Shain
[25] method because the oxidation process of the coordinated HNO was not clearly observed,
even at 5°C and a high scan rate (2 V s-1), which indicates that k-HNO >> k-NO for
trans-[Ru(NO)(NH3)4P(O-)(OEt)2](PF6)2 complex.
The HNO dimerizes to N2O (kdim = 8 × 106 M-1 s-1 [41]) in aqueous medium. Thus the
N2O identification [40] (Ecp = -0.32 V at pH 5.0 [42]) in solution, after one electron reduction
of trans-[Ru(NO)(NH3)4P(O-)(OEt)2]+ , can be a good indication of the HNO presence The
Fig. 2 shows the differential pulse voltammetric experiments carried out using a N2O
saturated solution and a solution containing trans-[Ru(NO)(NH3)4P(O-)(OEt)2]2+ at 2.5
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0.1°C and pH 5.0 (the N2O detection is pH dependent) before and after electrode polarization
at -1.0 V. The Ecp = -0.32 V observed in N2O saturated solution is similar to the one observed
in the solution containing trans-[Ru(NO)(NH3)4P(O-)(OEt)2]2+ indicating the release of HNO
in solution and its consequent dimerization into N2O.
Insert Figure 2
3.2 Aqueous medium stability of trans-[Ru(NO)(NH3)4P(O-)(OEt)2]2+
Trans-[Ru(NO)(NH3)4P(O-)(OEt)2]2+ was monitored at pH 3.0 using 31P NMR. The
31P NMR spectra initially exhibited just one peak at 68 ppm, which decayed (reaction 4) with
k = 8.90 × 10-7 s-1 (t1/2 = 9 days), yielding as the main product free diethyl phosphite and its
hydrolysed products (15-0 ppm), which were formed with t1/2 = 8.6 days.
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Two peaks of very low intensity at 66 and 62 ppm were also observed, corresponding
to trans-[Ru(NO)(NH3)4P(OH)2(OEt)]3+ and trans-[Ru(NO)(NH3)4P(O-)(OH)2]2+, respectively.
However, as judged from the product distribution analysis, the main reaction is the diethyl
phosphite dissociation (reaction 4) and not the phosphite ligand hydrolysis.
According to 31P NMR kinetic data obtained at pH 3.0 and 25
0.1°C,
trans-[Ru(NO)(NH3)4P(O-)(OEt)2]2+ (t1/2 = 9 days) is at least 3 times more stable than
trans-[Ru(NO)(NH3)4P(OEt)3]3+ (t1/2 = 2.8 days) [43] and 269 times more stable than
trans-[Ru(NO)(NH3)4P(O-)(OH)2]2+ (t1/2 = 47.7 min) [20].
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However, the behaviour of trans-[Ru(NO)(NH3)4P(O-)(OEt)2]2+ in aqueous media
does not match that observed for the corresponding nitrosyl complexes of triethyl phosphite
[43] or phosphorous acid [20] under the same experimental conditions.
Trans-[Ru(NO)(NH3)4P(OEt)3]3+ undergoes phosphorous ligand hydrolysis, whereas the
trans-[Ru(NO)(NH3)4P(O-)(OH)2]2+
isomerizes
to
the
O-bonded
compound
(trans-[Ru(NO)(NH3)4(O)P(OH)2]2+) and only then undergoes phosphorus ligand dissociation
[20].
On
the
other
hand,
the
diethyl
phosphite
in
trans-[Ru(NO)(NH3)4P(O-)(OEt)2]2+ neither hydrolyses nor isomerizes but dissociates as is.
This probably occurs due to the combination of the σ-inductive effect of the ethyl groups,
which are absent in phosphorous acid, and the dissociable proton, which is absent in triethyl
phosphite, which results in an extra negative charge over the phosphorus ligand, decreasing
the hydrolysis and isomerization reaction rates and favouring the dissociation.
Considering that trans-[Ru(NO)(NH3)4P(O-)(OEt)2]2+ is potentially useful as a
NO/HNO-donor in biological media, the stability of this complex was also monitored as a
function of time at pH 7.5 (TRIS buffer) by 31P NMR, IR and UV-vis spectroscopy as well as
cyclic voltammetry.
Fig. 3 shows the time course of the 31P NMR chemical shift areas for
trans-[Ru(NO)(NH3)4P(O-)(OEt)2]2+ at pH 7.5 and 25
0.5°C. The decay and formation
profile shown in Fig. 3 is not trivial, indicating that consecutive and/or competitive reactions
are taking place.
Insert Figure 3
Assuming first-order kinetic behaviour, the observed rate constants (k) of the decays
and formations of each δ31P in Fig. 3 can be obtained by plotting ln (A-At) versus time (s).
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In the time range of 0 to 7,000 s, the trans-[Ru(NO)(NH3)4P(O-)(OEt)2]2+ peak at 68
ppm decays, yielding peaks at 15-0 ppm, corresponding to free diethyl phosphite and its
hydrolysed products (kf(15-0 ppm)total = 1.14 × 10-4 s-1), and a peak at 138 ppm (kf(138 ppm) = 7.50
× 10-5 s-1).
After 7,000 s, the peak at 138 ppm decays, yielding a peak at 136 ppm (kf(136 ppm)
=
7.45 × 10-5 s-1). This last peak also decays, yielding free diethyl phosphite and its hydrolysed
products (kf(15-0ppm)2 = 6.25 × 10-5 s-1).
After 7,000 s, only the species at 136 ppm is able to generate free diethyl phosphite in
solution, with kf(15-0ppm)2 = 6.25 × 10-5 s-1. However, within the first 7,000 s, free diethyl
phosphite is produced at a rate of kf(15-0ppm)total = 1.14 × 10-4 s-1, indicating that during this
period, free diethyl phosphite is produced not only from the species at 136 ppm but also from
the initial species at 68 ppm. Thus, the rate of the dissociation of diethyl phosphite from
trans-[Ru(NO)(NH3)4P(O-)(OEt)2]2+ can be estimated as kf(15-0ppm)1 = 5.15 × 10-5 s-1 from kf(15-
0ppm)1 = kf(15-0ppm)total - kf(15-0ppm)2
.
The peaks at 68 ppm and 15-0 ppm, as described above, are known to correspond to
trans-[Ru(NO)(NH3)4P(O-)(OEt)2]2+ and P(OH)(OEt)2 and its hydrolysed species,
respectively. This is not the case for the peaks at 138 and 136 ppm. To identify these species,
trans-[Ru(H2O)(NH3)4P(O-)(OEt)2](PF6) was synthesized, and the 31P NMR spectra of the pH
7.5 solution containing this complex were obtained.
The 31P NMR spectrum of trans-[Ru(H2O)(NH3)4P(O-)(OEt)2](PF6) at pH 7.5
exhibited just one peak at 138 ppm. This peak diminishes as a function of time, yielding the
peak at 136 ppm. Thus, the peak at 136 ppm probably corresponds to the complex product of
diethyl phosphite ligand hydrolysis: trans-[Ru(H2O)(NH3)4P(O-)(OH)(OEt)]+.
12

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293
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297
Therefore, in solution at pH 7.5, trans-[Ru(NO)(NH3)4P(O-)(OEt)2]2+ (δ31P = 68 ppm)
is consumed competitively, yielding free diethyl phosphite (δ31P = 15-0 ppm, reaction 5) and
trans-[Ru(H2O)(NH3)4P(O-)(OEt)2]+ (δ31P = 138 ppm, reaction 7). This last complex
undergoes hydrolysis, giving rise to trans-[Ru(H2O)(NH3)4P(O-)(OH)(OEt)]+ (δ31P = 136
ppm, reaction 8), which also decays, yielding free diethyl phosphite (δ31P = 15-0 ppm,
reaction 9) as follows:
298
299
300
301
302
303
304
To produce trans-[Ru(H2O)(NH3)4P(O-)(OEt)2]+ (δ31P
=
138 ppm) from
trans-[Ru(NO)(NH3)4P(O-)(OEt)2]2+, a reaction centred on the nitrosonium ligand must occur.
According to the literature [14, 16], at pH ≥ 5.0, the nitrosyl complex undergoes nucleophilic
attack on the NO+ group, yielding
a
nitrite complex. The formation of
trans-[Ru(NO2)(NH3)4P(III)]+ can be indirectly determined spectrophotometrically through
13

305
306
307
the aquation of the nitrite ligand using pyrazine as an auxiliary ligand (reactions 10 and 11)
[16].
308
309
310
311
312
313
314
315
316
317
318
319
320
321
Thus, the formation of the trans-[Ru(pz)(NH3)4P(O-)(OEt)2]+ product (λ = 400 nm
[37],
Fig.
4)
was
followed
in
solution
at
pH
7.5
containing
trans-[Ru(NO)(NH3)4P(O-)(OEt)2]2+ and an excess of pyrazine. The initial nitrosyl complex
was converted into trans-[Ru(pz)(NH3)4P(O-)(OEt)2]+, and the observed rate constant (k) for
nitrite aquation in trans-[Ru(NO2)(NH3)4P(O-)(OEt)2] at pH 7.5 and 25
0.1°C was
calculated as 1.19 × 10-4 s-1.
Insert Figure 4
The same pH 7.5 solution containing trans-[Ru(NO)(NH3)4P(O-)(OEt)2]2+ and an
excess of pyrazine at 25 0.1°C was also monitored by 31P NMR. According to the spectra
acquired as a function of time, trans-[Ru(NO)(NH3)4P(O-)(OEt)2]2+ (δ31P = 68 ppm) yields
free diethyl phosphite (δ31P = 15-0 ppm) and trans-[Ru(pz)(NH3)4P(O-)(OEt)2]+ (δ31P = 132
ppm) with kf(132ppm) = 1.18 × 10-4 s-1. This value is experimentally indistinguishable from that
calculated using spectrophotometric data (k = 1.19 × 10-4 s-1).
322
323
324
325
326
327
328
Trans-[Ru(NO2)(NH3)4P(O-)(OEt)2] species were not detected in the 31P NMR spectra
of pH 7.5 solutions containing trans-[Ru(NO)(NH3)4P(O-)(OEt)2]2+. This absence is probably
due to rapid nitrite complex consumption (reactions 7), which precludes
trans-[Ru(NO2)(NH3)4P(O-)(OEt)2] accumulation.
14

329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
351
352
The infrared spectra of trans-[Ru(NO)(NH3)4P(O-)(OEt)2]2+ at pH 7.5 exhibited a
decrease in νNO+ = 1887 cm-1 (Fig. 5S) as function of time, which is also consistent with
nitrosyl complex consumption.
The cyclic voltammograms of trans-[Ru(NO)(NH3)4P(O-)(OEt)2]2+ at pH 7.5 and 25
0.1°C initially exhibited a Ru(III)/Ru(II) couple at 0.15 V and the same reduction processes of
NO+/NO and NO/HNO (E = -0.50 and -0.80 V vs SCE). This observation indicates that this
complex retains its ability to be activated through reduction and acts as a potential NO/HNO-
donor at pH 7.5. With time, the current of these processes decreases, indicating again the
consumption of the nitrosyl species.
It is interesting to note that the Ru(III)/Ru(II) couple in the
trans-[Ru(H2O)(NH3)4P(O-)(OEt)2]2+/trans-[Ru(H2O)(NH3)4P(O-)(OEt)2]+ species is now (pH
7.5) shifted by +130 mV relative to the voltammogram obtained at pH 2.0 due to the diethyl
phosphite deprotonation. Similar behaviour was observed when the ligand was phosphorous
acid (P(OH)3) [20], in which case the potential shifted by +120 mV.
Regarding nucleophilic attack on phosphite and on the nitrosyl group, the data
collected suggest that trans-[Ru(NO)(NH3)4P(O-)(OEt)2]2+ (t1/2 = 1.5 h at pH 7.5 and 25 0.5
°C) is the most robust among the trans-[Ru(NO)(NH3)4P(III)]n+ complex ions studied to date
[16,
20,
43].
The
formation
of
trans-[Ru(pz)(NH3)4P(O-)(OEt)2]+
from
trans-[Ru(NO)(NH3)4P(O-)(OEt)2]2+ at pH 7.5 (k = 1.19 × 10-4 s-1) shows that the aquation
rate of the nitrite ligand in trans-[Ru(NO2)(NH3)4P(O-)(OEt)2] is almost 100 times slower
than that in trans-[Ru(NO2)(NH3)4P(OEt)3]+ (1.30 × 10-2 s-1) [44]. The increase in the stability
of nitrite complex favours the in vivo catalytic cycle previously proposed [6, 9], in which the
nitrite complex is converted into a nitrosyl complex [16], rebuilding the NO/HNO-donor
complex.
15

353
354
355
356
357
358
359
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361
362
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365
366
367
368
369
370
371
372
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374
375
376
377
Considering that there are several indications that NO molecules play an important
role in pain modulation and can induce analgesia [45], in vivo tests using
trans-[Ru(NO)(NH3)4P(O-)(OEt)2]2+ as a NO/HNO-donor are being carried out. Preliminary
results regarding anti-inflammatory effects indicate that 100 µmol kg-1
trans-[Ru(NO)(NH3)4P(O-)(OEt)2]2+ considerably reduces the intensity of pain perception
(hypernociception) in Swiss mice (Fig. 6S) [46].
4. Conclusions
Trans-[Ru(NO)(NH3)4P(O-)(OEt)2](PF6)2 is the first isolated example in the
trans-[Ru(NO)(NH3)4L]3+ series of a phosphorus ester of the type P(OH)(OR)2 being bound
to ruthenium center through the phosphorus atom.
The presence of both σ−inductor groups (P-O-R) and an acidic proton (P-O-H) bonded
to the oxygen of phosphorus(III) precludes the hydrolysis and isomerization reactions on
trans-[Ru(NO)(NH3)4P(O-)(OEt)2]2+. In addition to being important in its own right and to the
chemistry of phosphorous esters as ligands, the obtained information concerning how R/H
groups modulate the phosphorus(III) ligand in terms of nucleophilic attack and isomerization
reactions in the trans-[Ru(NO)(NH3)4]3+ fragment will be helpful for better tailoring
NO/HNO delivery systems.
Lastly, the data presented herein show that trans-[Ru(NO)(NH3)4P(O-)(OEt)2]2+ is a
fast NO/HNO-donor and is rather stable in aqueous media over a wide range of pH (pH 3.0 -
7.5). Thus, it is a potential alternative to the unstable trans-[Ru(NO)(NH3)4P(OEt)3]3+ and
trans-[Ru(NO)(NH3)4P(O-)(OH)2]2+ ion complexes.
Acknowledgments
The authors acknowledge FAPESP, CNPq and CAPES for financial support
16

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Figure Captions
469
470
471
472
473
Fig. 1. Trans-[Ru(NO)(NH3)4P(O)(OEt)2]2+ in aqueous solution at pH 2.0, µ = 0.1 mol L-1
CRu = 1.5 × 10-3 mol L-1, T = 5 0.1°C. (a) Cyclic voltammogram obtained at a scan rate of
100 mV s-1; (b) differential pulse voltammogram obtained at a scan rate of 10 mV s-1.
,
474
475
476
477
478
Fig. 2. Differential pulse voltammograms at pH 5.0, µ = 0.1 mol L-1, T = 2.5 0.1°C and scan
rate of 10 mV s-1. Dotted line: DPV of trans-[Ru(NO)(NH3)4P(O-)(OEt)2]2+ at CRu = 3.0 × 10-3
mol L-1. Solid line: DPV of trans-[Ru(NO)(NH3)4P(O-)(OEt)2]2+ at CRu = 3.0 × 10-3 mol L-1
after electrode polarization at -1.0 V. Dashed line: DPV of N2O saturated solution.
479
480
481
482
483
484
485
Fig. 3. Product distribution as a function of time obtained from 31P NMR kinetics data for
trans-[Ru(NO)(NH3)4P(O)(OEt)2]2+ at pH 7.5 and 25 0.5°C.
(a) δ31P = 68 ppm; trans-[Ru(NO)(NH3)4(P(O)(OEt)2)]2+;
(b) δ31P = 138 ppm; trans-[Ru(OH)(NH3)4(P(O)(OEt)2)];
(c) δ31P = 136 ppm; trans-[Ru(OH)(NH3)4(P(O)(OH)(OEt))];
(d) δ31P = 15-0 ppm; P(OH)(OEt)2 and its hydrolysed forms.
486
487
Fig. 4. Electronic spectra of trans-[Ru(NO)(NH3)4P(O)(OEt)2]2+ in an excess of pyrazine at
pH 7.5 and 25 0.1°C as a function of time. Inserted: ln(A-At) versus time (s); R2 = 0.997.
488
489
19

490
491
492
493
Figure 1
494
495
496
497
498
499
500
501
502
503
504
505
506
507
508
509
510
511
512
20

513
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515
Figure 2
516
517
21

518
519
520
Figure 3
521
522
523
22

524
525
526
527
Figure 4
528
529
530
531
532
533
534
535
536
23

537
538
Table 1. NMR chemical shifts for trans-[Ru(NO)(NH3)4P(O)(OEt)2]2+ in D2O using the
TMSP-D4 and NH4PF6 as internal references.
Nuclei
31P
13C
Chemical Shift (ppm)
68.0
65.3
18.8
4.14
1.30
CH2
CH3
CH2
CH3
1H
539
540
541
24

542
543
Graphical Abstract
544
545
Graphical Abstract
546
547
548
549
550
551
The synthesis and the reactivity in aqueous media of the new compound trans-
[Ru(NO)(NH3)4P(O)(OEt)2](PF6)2 are described. This compound
is a potentially good NO/HNO-donor in biological media due to its remarkable robustness
over a wide pH range and exhibits a fast NO/HNO release when activated through one or two
electrons reduction. Preliminary in vivo tests indicate that this complex reduces pain
perception
552
25

553
554
555
Highlights
• The reactivity of the new trans-[Ru(NO)(NH3)4P(O)(OEt)2](PF6)2 in aqueous medium
is reported.
556
557
558
559
560
• This compound is a potential NO/HNO-donor upon electrochemical activation.
• It is stable in aqueous media over a wide pH range.
•
Trans-[Ru(NO)(NH3)4P(O)(OEt)2]2+ decays with a half-life of 1.5 h at pH 7.5 and
25°C.
561
562
26