Nitric oxide and nitroxyl products from the reaction of L -cysteine with trans-[RuNO(NH3)4P(OEt)3](PF6)3

Article abstract of DOI:10.1002/ejic.201402992

The reaction between the trans-[RuNO(NH₃)₄P(OEt)₃](PF₆)₃ and L-cysteine (RS^-) was studied over a pH range of 2.0-7.4. In this reaction, the concentrations of NO and HNO produced varied as a function of the pH of the solution. The first step of this reaction proceeded quickly [k₁ = (3.5 ± 0.3) × 10³ M^-1 s^-1, pH = 3.5, 25 C] and resulted in the formation of trans-[Ru(NH₃)₄P(OEt)₃N(O)SR]²⁺, which dissociated to yield trans-[Ru(NH₃)₄P(OEt)₃NO^·]²⁺ and RS^·. However, trans-[Ru(NH₃)₄P(OEt)₃N(O)SR]^n-1 can react with a second L-cysteine, yielding trans-[Ru(NH₃)₄P(OEt)₃N(O)(SR)₂]⁺ [k₂ = (3.6 ± 0.1) M^-1 s^-1, pH = 3.5, 25 C]. Therefore, the trans-[Ru(NH₃)₄P(OEt)₃NO^·]²⁺ species released NO and the trans-[Ru(NH₃)₄P(OEt)₃N(O)(SR)₂]^n-2 species released HNO.

Full text of DOI:10.1002/ejic.201402992

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DOI:10.1002/ejic.201402992
Nitric Oxide and Nitroxyl Products from the Reaction of
L
-Cysteine with trans-[RuNO(NH ) P(OEt) ](PF )
3
4
3
6 3
[a,b]
[a]
José Clayston Melo Pereira,
Douglas Wagner Franco*[
Maykon Lima Souza, and
a]
Keywords: Nitric oxide / Nitroxyl / Nitrogen oxides / Ruthenium / Kinetics / Medicinal chemistry
2+
and RS·. However, trans-
-cyste-
[k
s , pH = 3.5, 25 °C]. Therefore, the trans-
The reaction between the trans-[RuNO(NH
3
)
4
P(OEt)
3
](PF
6
)
3
trans-[Ru(NH
3
)
4
P(OEt)
3
NO·]
–
n–1
and
L-cysteine (RS ) was studied over a pH range of 2.0–7.4.
[Ru(NH P(OEt) N(O)SR] can react with a second L
3
)
4
3
+
In this reaction, the concentrations of NO and HNO produced
varied as a function of the pH of the solution. The first step
ine, yielding trans-[Ru(NH
3
)
4
P(OEt)
3
N(O)(SR)
2
]
2
=
–
1 –1
(3.6Ϯ0.1)
M
= (3.5Ϯ0.3)ϫ103
M
–1
s ,
–1
[Ru(NH
[Ru(NH
)
)
P(OEt)
P(OEt)
NO ] species released NO and the trans-
· 2+
of this reaction proceeded quickly [k
1
3
4
3
n–2
pH = 3.5, 25 °C] and resulted in the formation of trans-
3
4
3
N(O)(SR)
2
]
species released HNO.
2
+
[
3 4 3
Ru(NH ) P(OEt) N(O)SR] , which dissociated to yield
Introduction
NO or HNO electrochemically or through the chemical re-
+
[12,13]
duction of the nitrosonium ligand (NO ).
The reaction
Nitric oxide (NO) plays fundamental roles in biological
between trans-[RuNO(NH ) {P(III)} ](PF ) and euro-
3
4
3
6 3
processes such as immune responses, blood-pressure con-
pium(II), which is a one-electron reductant, quantitatively
trol, neurotransmission and carcinogenesis.[
1,2]
Nitroxyl
[12]
yields NO.
RuNO(NH ) {P(III)} ](PF ) complex and Zn(Hg) (a two-
In addition, the reaction between the trans-
(HNO), the product of nitric oxide reduction, has been re-
[
3
4
3
6 3
ported to be a more effective vasodilator than NO and to
exhibit a protective effect against heart failure. This finding
suggests that the HNO donors are a promising new class of
[12,13]
electron reductant) quantitatively yields HNO.
The
antileishmanial and antitrypanosomal effects of the follow-
ing complexes were ascribed to their nitric oxide and
nitroxyl release capacities when activated by one or two
electron reductions, respectively: trans-[Ru(NO)(NH ) L]-
[
3–7]
vasodilators.
The principal targets for NO under bioregulated condi-
tions are metal centers with low oxidation states, primarily
3
4
–
–
–
X , where X = BF , PF or Cl and L = imidazole (imN),
3
4
6
[
8]
iron proteins. In general, HNO interacts preferentially
with thiols and ferric hemes, which are intermediates in the
catalytic cycles of heme-based nitrite and nitric oxide re-
4-picoline (4-pic), pyrazine (pz), pyridine (py), triethylphos-
phite [P(OEt) ], l-histidine (l-hist), isonicotinamide (isn),
3
–2
sulfite (SO3 ) or nicotinamide (nic), see Equations (1) and
[
8]
ductases. Therefore, both NO and HNO molecules exhibit
relevant biological functions.[8–10] Focus has been placed on
the biological processes of the interactions of these nitric
oxide species with a metal center and on their redox in-
[10,12–15]
(2).
+
–
[11]
terconversions [M(NO )/M(NO) or M(NO )/M(HNO)].
This chemical knowledge was obtained through the synthe-
(1)
[
11]
sis and characterization of nitric oxide complexes.
For
species
where P(III) = P(OEt) or P(OH)(OEt) ] selectively releases
3
+
example, the trans-[Ru(NO)(NH ) {P(III)} ]
[
3
4
3
3
2
[
[
a] Instituto de Química de São Carlos, Universidade de São
Paulo,
(
2)
Av. Trabalhador São Carlense, CEP: 13560-970 – São Carlos –
SP, Brazil
Thiols are abundant in biological media and their redox
potentials are adequate for reactions with the nitrosonium
ligand. Thiols are therefore natural candidates for activa-
ting nitrosyl complexes in vivo. Indeed, in a related pa-
evidence was presented indicating l-cysteine reacts
with trans-[Ru(NO)(NH ) P(OEt) ] to yield final prod-
ucts of NO and HNO. To gain additional insight into the
E-mail: douglas@iqsc.usp.br
www.iqsc.usp.br
b] Departamento de Química Geral e Inorgânica, Instituto de
Química de Araraquara, UNESP – Universidade Estadual
Paulista,
[16]
[10]
per,
P. O. Box 355, Araraquara, São Paulo 14801-970, Brazil
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejic.201402992.
3+
3 4
3
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chemical reactivity of nitrosyl complexes with thiols, we
here investigated the reaction between trans-[Ru(NO)-
3
+
(
NH ) P(OEt) ] and l-cysteine.
3 4 3
Results and Discussion
As previously reported,[17–20] the reactions between ni-
trosyl ruthenium complexes and nucleophilic agents, such
as cysteine and glutathione, occur according to the reaction
sequences shown in Equations (3) and (4).
(
3)
Figure 1. Vibrational spectrum of the mixture of trans-[RuNO-
(
N
1
NH
O. Conditions: [Ru]
.2ϫ10 molL , pH 4.5, μ = 0.2 molL .
3 4 3 6 3
) P(OEt) ](PF ) and l-cysteine. Insert: peaks ascribed to
–
1
–1
2
=
1ϫ10 molL , [cys]tot
=
–
1
–1
–1
(
4)
n–1
Here, [Ru(L) N(O)SR]
is produced through interac-
tions between the thiol and the nitrosonium ligand. The
interactions of a second thiol molecule with [Ru(L) N-
5
The IR spectrum of the solution containing trans-[Ru-
3+
(
NO)(NH ) P(OEt) ] and cysteine displayed absorbance
3 4 3
–1
5
increases at 2236 and 2209 cm , which were attributed to
n–1
(
O)SR]
SR)2]
may result in the formation of [Ru(L) N(O)-
[Equation (3) and Equation (4)].
The production of the aqua ruthenium complex, cystine
RSSR), and N O or NO occurs in the sequences de-
[19,21]
5
the presence of N O.
Because N O can be a product
2
2
6
n–2
(
–1 –1
of HNO dimerization (k = 8.0ϫ10 m s ) and because
d
of the high concentration of the reagents, this observation
strongly suggests that nitroxyl could be formed from this
[
18]
[20]
(
2
picted in Equations (5) and (6).
[12,21]
reaction.
According to the chronoamperometric data, which were
corroborated by nitric oxide analyzer (NOA) measurements
(Figure S1, Supporting Information), the formation of NO
from the above reactions is pH dependent (Figure 2). The
5) concentration of liberated NO increased when the pH was
increased from 2.0 to 4.0 and decreased when the pH was
increased beyond 4.0.
(
(
6)
Therefore, the reaction between the trans-[Ru(NH ) P-
3
4
3
+
(
OEt) NO] complex and cysteine would likely follow a
3
[18–20]
pathway similar to those previously mentioned
[Equa-
tion (5) and Equation (6)]. To verify these hypotheses, we
conducted kinetic studies and product analyses for these
reactions.
Production of NO and HNO from the Reaction between the
Nitrosyl Complex and L-Cysteine
Figure 2. Nitric oxide formation in the reaction between trans-
3
+
[Ru(NH
3 4 3
) P(OEt) NO] and l-cysteine (at two different concentra-
The reaction was monitored by using infrared spec-
troscopy and chronoamperometry. After mixing aqueous
solutions of the complex trans-[Ru(NH ) P(OEt) NO] (1)
tions) as a function of the solution pH. Experimental conditions:
–
4
–1
–3
–1
[
Ru] = 5ϫ10 molL , ᭿ [cys]tot = 5ϫ10 molL , ᮀ [cys]tot =
3
+
–2
–1
–
3
4
3
tot
5ϫ10 molL ; [cys] = ([RS ] + [RSH]), T = 25 °C.
and l-cysteine, we detected free nitric oxide as indicated by
–
1
an increase in the IR absorbance at 1872 cm ( ν˜ NO in
Assuming that the reaction between cysteine and the
0
RuNO ) with a simultaneous decrease in the absorbance nitrosyl complex involves a one-electron reduction of the
–
1
+[17]
3+
at 1923 cm , which corresponds to the ν˜
NO+
in RuNO
trans-[Ru(NH ) P(OEt) NO] species, only 6% of the total
3 4 3
(Figure 1).
NO expected to form was observed at pH 7.4. In contrast,
Eur. J. Inorg. Chem. 0000, 0–0
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when Na [Fe(CN) ] was simultaneously added to the solu-
3
6
tion (pH 7.4) with the trans-[RuNO(NH ) P(OEt) ](PF ) ,
3
4
3
6 3
(10)
the amount of NO formed reached 36% of the expected
[
10]
value. This increase in the NO concentration is consistent
with the presence of HNO in the medium, which likely re-
we calculated
a
rate constant of
k1(RS)tot
=
=
3
–1
–1
3
–
(3.5Ϯ0.3)ϫ10 mol Ls
and
k–1(RS)tot
acted with [Fe(CN) ] to generate the additional nitric ox-
6
1
–1
ide[ through the following reaction [Equation (7)].
22]
(4.7Ϯ0.5)ϫ10 s at pH 3.5 (Figure S2, Supporing Infor-
mation).
(
7)
In addition, Figure 2 illustrates the effect on the pro-
duction of NO by this system upon increasing the l-cyste-
ine concentration. Notably, as the [RS] was increased
tot
–
3
–2
from 5ϫ10 to 5ϫ10 m in the presence of the ruthenium
–
4
complex (5ϫ10 m), the amount of NO produced in-
creased in the pH range of 2.0–4.0 reaching a maximum
value at pH 4.0 and which significantly decreased at pH
values higher than 4.0. Given that the pK of cysteine is
a
[
23]
8.3,
this behavior is consistent with an increase in the
concentration of deprotonated l-cysteine, which inhibits
NO production.
2
+
Formation of trans-[Ru(NH ) P(OEt) N(O)SR] Species
3
4
3
The reaction of l-cysteine with trans-[Ru(NH ) P-
3
4
3
+
(
OEt) NO] was monitored on the basis of the changes in
3
the electronic spectra using stopped-flow and conventional
techniques (Figure 3, A and B). The initial spectrum of the
solution underwent a change in the millisecond timescale
3
+
upon reaction of trans-[Ru(NH ) P(OEt) NO] with cyste-
3
4
3
ine. An increase in the absorbances at 316 and 465 nm was
noticed (Figure 3, B) with respect to the starting solution.
Figure 3. (A) spectroscopic change for the reaction solution of
On the basis of the experimental data and information
3+
trans-[Ru(NH
3
)
4
P(OEt)
3
NO]
and cysteine monitored by the
reported in the literature[
17–20,22]
for related systems, a nu- stopped-flow technique. Insert: absorbance as a function of the
time (s) for the reaction monitored using the stopped-flow tech-
cleophilic attack on the nitrogen by a thiol sulfur atom,
such as in the reactions illustrated in Equations (8) and (9)
is anticipated.
3
+ __
nique. (B) Electronic spectra of trans-[Ru(NH
3
)
4
P(OEt)
3
NO] ( )
P-
3
OEt) NO] and l-cysteine (---). Experimental conditions: [Ru] =
and of the products of the reaction between trans-[Ru(NH )
3 4
3+
(
5
0
–
4
–1
–3
–1
ϫ10 molL , [cys]tot = 5ϫ10 molL , pH = 3.5, μ =
–
1
.2 molL , T = 25 °C.
+
Formation of trans-[Ru(NH ) P(OEt) N(O)(SR) ] Species
3
4
3
2
(
8)
+
The formation of trans-[Ru(NH ) P(OEt) N(O)(SR) ]
3
4
3
2
occurs in the slower second step (Table 1) of the reaction in
a matter of seconds, resulting in an absorbance decrease at
Table 1. Kinetic results for the reaction between L-cysteine and
3
+
trans-[Ru(NH
3
)
4
P(OEt)
3
NO] .
Step 1
k
1
[m–1 s–1]
Step 2
k
2
[m–1 s–1]
(
9)
RS)total[
a]
3.5ϫ10
1.4ϫ10
3
8
(RS)total
[a]
3.6
1.6ϫ10
(
–
–
4
On the basis of the changes in absorbance (Figure 3, A), (RS )
(RS )
–
the assumption that [RS]tot = [RS ] + [RSH] and the expres-
sion in Equation (10)
–
[a] pH 3.5 (0.1 mm, acetate buffer), T = 25 °C, [RS]tot = [RS ] +
[RSH].
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(
(
13)
14)
When a pK of 8.3 is considered for the reaction of RSH
a
–
+
+
h RS + H and when [H ] ϾϾ K , a plot of the second-
a
order rate constant k against 1/[H] should be linear, with
2
the slope and intercept equal to k2
(RS–)
·Ka and k2(RSH),
[18]
respectively (Figure 4, B), as suggested by Rocaroli.
Thus, the rate constant k2(RS–) was calculated as
1.6Ϯ0.2)ϫ10 m s and the intercept was k–
4
–1 –1
(
(
2(RS–)
=
–1
3.1Ϯ0.2) s .
By extrapolating the data from Equation (13) for the first
step of the reaction, we calculated k values at pH 3.0 and
1
.5 as approximately 1.14ϫ108 and 1.76ϫ10 m s ,
8
–1 –1
3
respectively. Thus the estimated average k would be ap-
1
8
–1 –1
proximately (1.4Ϯ0.3)ϫ10 m s , which indicates that
the first reaction step was much faster than the second reac-
tion step (Table 1).
n
Degradation of trans-[Ru(NH ) P(OEt) N(O)SR] and
3
4
3
n–2
Figure 4. (A) Electronic spectra of trans-[Ru(NH
3
)
4
P(OEt)
3
N(O)-
] (---). Insert: ab-
sorbance vs. time (s). Experimental conditions: [Ru]
trans-[Ru(NH ) P(OEt) N(O)(SR) ] Species
3
4
3
2
SR]2
+
(
___
+
3 4 3 2
) and trans-[Ru(NH ) P(OEt) N(O)SR
=
With time, an additional decrease was observed in the
absorbance at 320 nm (on a minute timescale) as the maxi-
mum simultaneously shifted to 316 nm (Figure 5). The final
5
0
ϫ10–4 molL , [cys]tot = 5ϫ10 molL , pH = 4.5, μ =
–1
–3
–1
–1
2
.2 molL , T = 25 °C. (B) Values of k vs. pH for the reaction of
3 4 3
) P(OEt)
NO]3 and l-cysteine. μ = 0.2 molL , T
+
–1
trans-[Ru(NH
25 °C.
solution spectrum corresponds to the trans-[Ru(NH ) P-
=
3 4
2
+
[24]
(
OEt) H O] ion.
On the basis of these last spectro-
3
2
–
4 –1
scopic changes, a value of kobs = 4.9ϫ10 s was calcu-
lated. At first glance, we associated the spectroscopic
change with degradation of trans-[Ru(NH ) P(OEt) N-
465 nm and a simultaneous increase in the solution ab-
sorbance at 320 nm (Figure 4, A).
The k2 values calculated from these spectroscopic
changes (Figure 4, B) were pH dependent, as show in Equa-
3
4
3
2+
(O)(SR)]
– see Equations (15) and (16) – or trans-
+
[
Ru(NH ) P(OEt) N(O)(SR) ] .
3
4
3
2
tions (11) and (12) and were calculated as k
=
2
(RS)tot
–
1 –1
–3 –1
(3.6Ϯ0.1) m s and k–2(RS)tot = (3.8Ϯ0.8)ϫ10 s at pH
3
.5 (Figure S3, Supporting Information) when considering
–
the total [RS]tot = ([RS ] + [RSH]).
(
(
15)
16)
(
(
11)
12)
The NO production increased as the trans-[Ru(NH ) P-
3
4
2+
(
OEt) N(O)SR] concentration increased until it reached
3
a maximum at pH 4.0 and [Cys]tot = 5ϫ10–3 molL (Fig-
–1
ure 2). Thus, under these experimental conditions, most of
the trans-[Ru(NH ) P(OEt) N(O)SR] ion would generate
2+
3
4
3
2+
·
the trans-[Ru(NH ) P(OEt) H O] , NO and RS ; see
3
4
3
2
According to the literature,[18] this step of the reaction Equation (15). Indeed, the trans-[Ru(NH ) P(OEt) H O]
2+
3
4
3
2
between nitrosyl ruthenium complexes and a thiol can be ion was identified by using UV/Vis spectroscopy (Figure 4,
expressed as shown in Equations (13) and (14).
B) and 31P NMR spectroscopy (δ = 148 ppm, Figure S4,
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but with increasing cysteine concentrations (Figure 2). This
behavior is consistent with the equilibrium constants as
shown below in Equations (18), (19), (20) and (21).
(
(
18)
19)
(
(
20)
21)
Figure 5. Electronic spectra of the trans-[Ru(NH
SR] and trans-[Ru(NH ) P(OEt) N(O)SR ]
3 4 3 2
3
)
4
P(OEt)
decomposition. In-
sert: absorbance vs. time(s). Experimental conditions: [Ru] =
3
N(O)-
3
+
2+
–1
–3
–1
5
0
ϫ10–4 molL , [cys]tot = 5ϫ10 molL , pH = 4.5, μ =
–1
.2 molL , T = 25 °C.
According to Equation (19) and Equation (21), an in-
crease in the solution pH would favor the formation of the
trans-[Ru(NH ) P(OEt) N(O)(SR) ] species over the trans-
+
3
4
3
2
Supporting Information).[24] However, in a large concentra- [Ru(NH
) P(OEt) N(O)SR] species. Therefore, high pH
2+
3 4 3
tion of cysteine or at pH greater than 4.0, the concentration would induce high HNO production.
–
of RS increases and the NO production decreases because
of the likely formation of the trans-[Ru(NH ) P(OEt) -
3
4
3
trans Effect and trans Influence of P(OEt)3
+
N(O)(SR) ] ion according to Equation (11) and Equation
2
Because of the trans effect and trans influence of the
(
12). This species is suggested to lead the reduction of the
[
24]
+
P(OEt) ligand,
[
the dissociation of NO from the trans-
NO ligand by two electrons producing HNO as shown in
Equation (16). Thus, under these conditions, production of
HNO molecules can be favored. Indeed, HNO has been
3
+
II
Ru(NH ) P(OEt) N(O)] species (in which the Ru –NO
3 4 3
[
26]
backbonding is very weak)
may be faster than in the
n
ruthenium nitrosyl complexes [Ru(L) MNO] (L = poly-
detected indirectly through the presence of N O (ν˜
=
5
18]
2
2
N O
[
–
1
pyridines, NH , EDTA, pz and py).
The relatively slow
2
236 and 2209 cm ) and by the oxidation of HNO to NO
3
3
–
[10]
dissociation of NO from other ruthenium ammines i.e.
during the reaction with [Fe(CN)6] [Equation (7)]. Ad-
ditional support for HNO production in the reaction be-
3
+
trans-[RuNO(NH ) (L)] , such as L = pyrazine [k
=
NO
could facili-
3 4
–1
0
[26,27]
3
+
0
.07 s , E = 0.112 V vs. NHE]
+
0
tween trans-[Ru(NO)(NH ) P(OEt) ]
and cysteine was
(NO /NO )
3
4
3
tate the second nucleophilic attack – see Equations (11) and
provided in the literature where metmyoglobin was reported
as the nitroxyl scavenger at pH 7.4.[
10,25]
Therefore, both
(12) – resulting in the formation of HNO and reducing the
0
Ј
amount of NO formed. Furthermore because the E
NO and HNO can be produced from the reaction between
3+
2+
3+
3
+
[
RuNO] /[RuNO] in the trans-[Ru(NH ) P(OEt) NO]
trans-[Ru(NO)(NH ) P(OEt) ] and cysteine owing to the
3 4 3
3
4
3
[
24]
2
+
complex is more positive (+0.132 V vs. NHE)
than that
formation of trans-[Ru(NH ) P(OEt) N(O)SR] [Equation
3
4
3
+
in the other tetraammines, the nitrosyl ligand in this com-
pound would be sufficiently active to undergo nucleophilic
attack by cysteine even at pHϽ3. Scheme 1 illustrates a
(
(
15)] and trans-[Ru(NH ) P(OEt) N(O)(SR) ] [Equation
3 4 3 2
16)], respectively, which occurs as a function of the concen-
tration of RS . The aqua complex trans-[Ru(NH ) -
P(OEt) (H O)] is the ruthenium based product formed
–
3
4
2
+
3
2
following the NO and HNO release. The pseudo-first-order
rate constant (kobs) for the formation of this aqua complex
can be expressed as shown in Equation (17), which shows
2+
dependence on the trans-[Ru(NH ) P(OEt) N(O)SR] and
3
4
3
+
trans-[Ru(NH ) P(OEt) N(O)(SR) ] concentrations.
3
4
3
2
(
17)
As has been suggested, the increased formation of the
+
trans-[Ru(NH ) P(OEt) N(O)(SR) ] ion was associated
3
4
3
2
with decreased NO production. This was identified by con-
NO]3+ and l-
3 4 3
) P(OEt)
Scheme 1. Reaction between trans-[Ru(NH
ducting the reaction at the same pH and [RS ]/[RSH] ratio cysteine.
–
Eur. J. Inorg. Chem. 0000, 0–0
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potential mechanism for the reaction between trans- CH
3
COONa or C
buffer solutions and the ionic strength was held constant by using
NaCF COO as a supporting electrolyte. The observed pseudo-first-
order constants (obs)were determined from plots of ln(A – A ) vs.
time. Following treatment of the data according to kobs = k [L] + k–1
3 5 3 3 5 3 3
H O(COOH) /C H O(COO) Na were used as
3
+
[
Ru(NH ) P(OEt) NO] and l-cysteine.
3 4 3
3
ϱ
t
1
,
Conclusions
–
where L = RSH or RS , the specific second-order rate constants
were calculated according to the equilibrium equations for the re-
spective reactions. The amount of NO that was released in these
solutions was measured by using a selective NO electrode (amino)
from Innovative Instruments, Inc. The temperature was maintained
at T = 25.0Ϯ0.1 °C. The experimental conditions were held con-
stant except for the concentration of l-cysteine, which was varied
Evidence for the formation of both HNO and NO spe-
cies was collected for the reaction between l-cysteine and
the trans-[Ru(NH ) P(OEt) NO]
product distribution was found to depend on the RSH/RS
ratio in solution.
3
+
ion. In addition, the
3
4
3
–
The release of HNO was favored when the trans- from 10 to 100 times greater than the concentration of the nitrosyl
3
+
[
Ru(NH ) P(OEt) NO] complex reacted with l-cysteine complex. The trans-[Ru(NO)(NH ) P(OEt) ](PF ) compound
3 4 3
3 4 3 6 3
–
at pH 7.4. In this case, the RS concentration was percepti- (4.0 mg, 500 μm) was dissolved in 10 mL of an Ar-saturated solu-
ble. In macrophage and tumor cells and under metabolic
conditions such as hypoxia, where the pH is slightly acidic
in relation to normal cells,
tion containing l-cysteine. The experiments were performed over a
pH range of 2.0 to 7.4 at 25 °C. The model 280i nitric oxide ana-
lyzer (NOA) from the GE Sievers was also used in the NO measure-
ments at pH 4.0 and 7.4.[
[
28,29]
compounds like trans-
33]
3
+
[
Ru(NH ) P(OEt) NO] would work as sources of NO.
3
4
3
Acknowledgments
Experimental Section
Chemicals and Reagents: All chemicals (unless otherwise indicated)
were analytical grade and purchased from Aldrich, Strem or Sigma.
Ruthenium trichloride hydrate was the starting material for the syn-
thesis of all of the ruthenium complexes described herein. All sol-
The authors acknowledge the Brazilian agencies Fundação de Am-
paro à Pesquisa do Estado de São Paulo (FAPESP), Coordenação
de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and
Conselho Nacional de Desenvolvimento Científico e Tecnológico
(CNPq) for their financial support. The authors also thank José C.
Toledo Jr. (FFCLRP-USP) for NOA measurements.
[30]
vents were purified following known procedures.
tilled water was used throughout the experiments.
Doubly dis-
Synthesis of the Complexes: All syntheses and manipulations were
atmosphere.[
31]
conducted
The[Ru(NH
under
Cl]Cl
an
argon-containing
[
[
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3
)
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2
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[18]
9
9.99%) was deoxygenated (Cr ) prior to use.
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[
[
[
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[
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© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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Pages: 8
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FULL PAPER
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Date: 28-01-15 13:25:22
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Model Complex for NO Release
J. C. M. Pereira, M. L. Souza,
D. W. Franco* ................................... 1–8
The concentration of nitric oxide and ni-
troxyl generated from the reaction between
trans-[RuNO(NH
3
)
4
P(OEt)
3
](PF
6
)
3
and
Nitric Oxide and Nitroxyl Products from
the Reaction of l-Cysteine with trans-[Ru-
cysteine can be modulated by altering the
–
pH. The nucleophilic attack of RS yielded
2
+
NO(NH
3
)
4
P(OEt)
3
](PF
6
)
3
mostly trans-[Ru(NH
3
)
4
P(OEt)
3
N(O)SR]
P(OEt)
at pH Ն 6.0, which decom-
at pH Յ 4.0 and trans-[Ru(NH
3
)
4
3
-
2
+
Keywords: Nitric oxide / Nitroxyl / Nitro-
gen oxides / Ruthenium / Kinetics / Med-
icinal chemistry
2
N(O)(SR) ]
pose into NO and HNO, respectively.
Eur. J. Inorg. Chem. 0000, 0–0
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