PAMAM dendrimers functionalized with ruthenium nitrosyl as nitric oxide carriers Metallodendrimers Special Issue

Article abstract of DOI:10.1016/j.ica.2013.07.009

The functionalization of three generations of polyamidoamine (PAMAM G0, G2 and G3) dendrimers with the NO-donor trans-[Ru(NO)(NH₃) ₄(ina)](BF₄)₃ (ina = isonicotinic acid) is reported. PAMAMs were modified through a peptide-type bond between the carboxyl group of the ina ligand and the dendrimer superficial amines. Compounds were characterized by FT-IR, UV-Vis, CV, DPV, ¹H NMR, ICP-OES, and the structure of the complextrans-[Ru(NO)(NH₃)₄(ina)](BF ₄)(SiF₆)·H₂O was determined by single crystal X-Ray analysis. The experimental data indicated the immobilization of 4, ～8 and ～27 nitrosyl complexes on the G0, G2 and G3 dendrimer's surface, respectively, which corresponds to ～1.0-1.43 μmol NO per mg of dendrimer. FT-IR, UV-Vis and electrochemical assays suggest that the functionalization of PAMAM did not alter the coordination sphere of the ruthenium nitrosyl complex neither the formal reduction potential of Ru ^IINO⁺/Ru^IINO⁰ couple regarding to the complex not attached to PAMAM. The NO release in these compounds, through light irradiation (λ = 355 nm) and one-electron reduction (Eu ²⁺), was investigated.

Full text of DOI:10.1016/j.ica.2013.07.009

Inorganica Chimica Acta 409 (2014) 147–155
Contents lists available at ScienceDirect
Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
PAMAM dendrimers functionalized with ruthenium nitrosyl as nitric
oxide carriers
Antonio Carlos Roveda Jr. a, Thiago Bueno Ruiz Papa a, Eduardo Ernesto Castellano b,
Douglas Wagner Franco a,
⇑
a Instituto de Química de São Carlos, Universidade de São Paulo, Av. Trabalhador São-carlense, n° 400, 13566-590 São Carlos, SP, Brazil
b Instituto de Física de São Carlos, Universidade de São Paulo, Av. Trabalhador São-carlense, n° 400, 13566-590 São Carlos, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 11 July 2013
The functionalization of three generations of polyamidoamine (PAMAM G0, G2 and G3) dendrimers with
the NO-donor trans-[Ru(NO)(NH3)4(ina)](BF4)3 (ina = isonicotinic acid) is reported. PAMAMs were modi-
fied through a peptide-type bond between the carboxyl group of the ina ligand and the dendrimer super-
ficial amines. Compounds were characterized by FT-IR, UV–Vis, CV, DPV, 1H NMR, ICP-OES, and the
structure of the complextrans-[Ru(NO)(NH3)4(ina)](BF4)(SiF6)ꢀH2O was determined by single crystal X-
Ray analysis. The experimental data indicated the immobilization of 4, ꢁ8 and ꢁ27 nitrosyl complexes
Metallodendrimers Special Issue
Keywords:
PAMAM dendrimer
Functionalization
Ruthenium nitrosyl complexes
Nitric oxide release
NO-donor
on the G0, G2 and G3 dendrimer’s surface, respectively, which corresponds to ꢁ1.0–1.43
lmol NO per
mg of dendrimer. FT-IR, UV–Vis and electrochemical assays suggest that the functionalization of PAMAM
did not alter the coordination sphere of the ruthenium nitrosyl complex neither the formal reduction
potential of RuIINO+/RuIINO0 couple regarding to the complex not attached to PAMAM. The NO release
in these compounds, through light irradiation (k = 355 nm) and one-electron reduction (Eu2+), was
investigated.
Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction
Similar to other NO donors, ruthenium nitrosyl complexes have
been attached to different platforms aiming diverse purposes
The free radical nitric oxide (NOꢀ) is an important signaling mol-
ecule [1,2], endogenously produced in the human body, which is
associated with several biological functions [3]. The wide range
of physiological and pathophysiological functions in which NO is
involved, demands methods for storage and control the NO deliv-
ery at specific sites. Different compounds have been developed as
effective NO donors, such as nitrosothiols, diazeniumdiolates, or-
ganic nitrates and nitrites, and also metal nitrosyl complexes
[4,5]. These compounds are helpful to better understand the bio-
logical functions of NO and some of them exhibited potential ther-
apeutic activities [6–11].
Among the different NO donors, ruthenium nitrosyl complexes
of the type trans-[Ru(NO)(NH3)4(L)]3+ [12] are particularly attrac-
tive due to their water solubility, low toxicity to the host cells
[7], stability in aqueous solution in the presence of oxygen [13]
and robustness regarding substitution reactions [14]. The NO re-
lease in these compounds can be triggered either by one-electron
reduction [12] or by light irradiation [15].
[16,17], such as the production of materials with a high NO pay-
load [18], the controlled NO bioavailability at specific sites [19]
and the improvement of NO donors stability [20]. Silica [21,22],
xerogels [23–25], liposomes [26,27], nanoparticles [28] and den-
drimers [18,29,30], are common examples of matrices that have
been used as NO-releasing vehicles [17,31]. Dendrimers are attrac-
tive molecules for NO transport, once they can provide a scaffold
for storing large amounts of NO on a single framework [18]. They
also have a well defined branching structure with a multivalent
surface, which can be useful for simultaneous interactions to mul-
tiple receptors [32,33]. These molecules have been extensively
studied as drug delivery systems and imaging contrast [34–36].
Among the variety of dendrimers, Polyamidoamine (PAMAM) is a
noteworthy class due to its water solubility and amine or carboxyl
surface, which allows tailoring through different kinds of reactions
with molecules or ions of interest [37–39].
Some dendrimers have already been synthesized aiming nitric
oxide transport and release [18,19,29,30,20]. For example Stasko
and Schoenfisch [18] synthesized polypropylenimine (PPI) dendri-
mers (G3 and G5) functionalized with N-diazeniumdiolates which
were able to store ꢁ5.6
lmol NO per mg of compound. Following a
similar approach, Lu et al. [20] synthesized a series of amine PPI
⇑
Corresponding author. Tel.: +55 16 3373 9970; fax: +55 16 3373 9976.
E-mail address: douglas@iqsc.usp.br (D.W. Franco).
dendrimers (G2–G5) functionalized with different surface groups,
0020-1693/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ica.2013.07.009

148
A.C. Roveda Jr. et al. / Inorganica Chimica Acta 409 (2014) 147–155
in which the NO storage capacity was in the range of 0.9–3.8
lmol
2.3. Measurements
NO per mg of compound [20]. Stasko and collaborators [29] also
synthesized a G4 PAMAM functionalized with S-nitrosothiols
All experiments were carried out at 25 1 °C in a phosphate
which were able to store ꢁ2
l
mol NO per mg of compound. Benini
buffer pH 7.4 (0.10 mol Lꢂ1), = 0.20 mol Lꢂ1, or in aqueous solu-
l
and collaborators [30] functionalized PAMAM dendrimers (G0, G2
tion pH 2.0, l
= 0.10 mol Lꢂ1 (CF3COOH/CF3COONa). All manipula-
and G3) with the [Ru(edta)(NO)]ꢂ complex, which were capable of
tions were performed in the absence of oxygen. The inert gas
(argon with high purity 99.998%) was deoxygenated by passing
through a Cr(II) solution prior to use [40]. The nitrogen gas
(99.999% of purity) was used without further purification. The
complexes were stored under vacuum and protected from light
and moisture. For ruthenium analysis, a calibration curve was pre-
pared using a commercial standard ruthenium solution (1000 mg/L
of Ru in HNO3 2% water solution). The samples were prepared dis-
solving the ruthenium complexes in a trifluoroacetic acid solution
(1.0 ꢃ 10ꢂ3 mol Lꢂ1) and aliquots were taken for the analysis (ICP-
OES). NMR, EPR and UV–Vis spectra of the solutions containing air-
sensitive complexes were obtained under argon atmosphere. Solu-
tions were transferred through Teflon tubing to specific cell or tube
using the inert gas pressure.
storing around 1.4–1.8 lmol NO per mg of compound.
In this context, this work reports the synthesis and character-
ization of the complex trans-[Ru(NO)(NH3)4(ina)]3+ (ina = isonicot-
inic acid) attached to PAMAM dendrimers of generations 0, 2 and 3.
Some reactivity aspects of these compounds regarding NO release
are also discussed. The ina ligand was chosen because it provides a
carboxyl group for the attachment to PAMAM’s superficial amines.
Furthermore, after the amide bond formation, the structure of the
complex trans-[Ru(NO)(NH3)4(ina)]3+ attached to the dendrimer
becomes similar to the structure of trans-[Ru(NO)(NH3)4(isn)]3+
(isn = isonicotinamide), which exhibited the best results among
the complexes of type trans-[Ru(NO)(NH3)4(L)]3+ in tests against
Chaga’s disease [7,8].
2.4. Synthesis of trans-[Ru(NO)(NH3)4(ina)](BF4)3
2. Experimental section
The complexes trans-[Ru(NH3)4(ina)(SO4)]Cl [41] and trans-
[Ru(NO)(NH3)4(ina)](BF4)3 were synthesized and characterized as
described in the literature [13,14]. Theoretical elemental analysis
for trans-[Ru(NH3)4(ina)(SO4)]Clꢀ2H2O: C, 15.67; H, 4.60; N, 15.23.
Experimental elemental analysis: C, 15.90; H, 4.68; N, 14.92. Yield:
65–70%. Theoretical elemental analysis for trans-[Ru(NO)(NH3)4(-
ina)](BF4)3ꢀ2H2O: C, 11.65; H, 3.42; N, 13.58. Experimental elemen-
tal analysis: C, 11.51; H, 3.52; N, 13.43. Yield: 55–60%.
2.1. Chemicals and reagents
The analytical grade reagents (Aldrich or Merck) and solvents
(Mallincrodt, Baker, Merck or Panreac) were used as purchased, ex-
cept PAMAM dendrimers (20 wt.% in methanol), which were dried
under vacuum to remove methanol before using. Ruthenium tri-
chloride (RuCl3ꢀxH2O) was the starting reagent for the synthesis
of all the complexes described herein. The synthesis and manipu-
lations were performed under an argon atmosphere using standard
techniques [40]. Eu2+ solution was prepared by adding Eu2O3
(99.99%) in a deaerated acid solution (0.10 mol Lꢂ1 trifluoroacetic
acid) containing Zn(Hg). The reduction of Eu3+ to Eu2+ was com-
pleted after 40 min, and then the solution was used immediately.
Deionized water (Millipore) was used throughout this work.
2.5. X-ray data collection and structure determination
Crystals of trans-[Ru(NO)(NH3)4(ina)](BF4)(SiF6)ꢀH2O were ob-
tained from an aqueous solution (4.0 mol Lꢂ1 of HBF4) of trans-
[Ru(NO)(NH3)4(ina)]3+ maintained at ꢁ10 °C for one week. The
presence of SiF26ꢂ as counterion in the crystals was originated from
the reagent HBF4, which contains SiF2ꢂ as impurity (ꢁ0.2%), as pre-
6
2.2. Instruments
viously reported [13]. The presence of SiF26ꢂ as counterion in the
crystals was also identified by FT-IR measurements through the
bands at 740 and 480 cmꢂ1 [42], which are absent in the amor-
phous solid trans-[Ru(NO)(NH3)4(ina)](BF4)3ꢀ2H2O. The band at
1080 cmꢂ1, assigned to BFꢂ4 anion [42], was also observed in the
FT-IR for the complex trans-[Ru(NO)(NH3)4(ina)](BF4)(SiF6)ꢀH2O. It
is important to emphasize that the amorphous solid of trans-
[Ru(NO)(NH3)4(ina)]3+ has three BFꢂ4 as counterions, as confirmed
by the FT-IR measurements and elemental analysis. Theoretical
elemental analysis for trans-[Ru(NO)(NH3)4(ina)](BF4)(SiF6)ꢀH2O:
C, 12.66; H, 3.36; N, 14.76. Experimental elemental analysis: C,
12.41; H, 3.53; N, 14.56.
Electronic spectra were recorded in a Hitachi U-3501 or Agilent
8453 UV–Vis spectrophotometer model using a 1.00 cm quartz
cell. The solid-state infrared measurements were recorded in a Bo-
mem MB 102 FTIR spectrophotometer using KBr pellets, 128 scans
and resolution of 4 cmꢂ1 in the 4000–400 cmꢂ1 range. The FTIR
measurements for PAMAM (oil) were performed in a silicon win-
dow using the same conditions described before for the assays
using KBr pellets. The elemental analyses were performed on a Per-
kin–Elmer CHN 2400. Ruthenium analysis was carried out using a
Perkin Elmer Optima 3000 DV Induced Coupled-Plasma Optical
Emission Spectrometer (ICP-OES). Cyclic voltammetry (CV) and dif-
ferential pulse voltammetry (DPV) experiments were performed
with a PAR model 264A Potentiostat. The three-electrode system,
saturated calomel, glassy carbon and platinum wire, were used
as reference, work and auxiliary electrodes respectively. The po-
tential values were converted and reported as normal hydrogen
electrode (NHE). 1H NMR spectra were recorded in a Bruker DRX
400 spectrometer using a trifluoroacetic acid-d solution (CF3COOD,
1 mol Lꢂ1) in D2O. 3-(Trimethylsilyl)propionic-2,2,3,3-d4 acid so-
dium salt was used as internal reference. Electron paramagnetic
resonance (EPR) spectra were obtained in a Bruker EMX Plus
X-band spectrometer at room or liquid nitrogen temperature.
Samples were irradiated with a Nd:YAG LASER (Continuum, model
Surelite-II) operating in the third harmonic (k = 355 nm). A power
meter (Coherent, Lasermate-P) measured the average energy per
pulse.
2.5.1. Crystal data
A yellow crystal of dimensions 0.163 ꢃ 0.036 ꢃ 0.026 mm3 was
selected and mounted on an Enraf–Nonius Kappa-CCD diffractom-
eter with graphite monochromated Mo K
a (k = 0.71073 Å) radia-
tion. Data were collected at room temperature up to 52° in 2h
and final unit cell parameters were based on all reflections.
2.5.2. Data collection and processing
Data collections were made using the COLLECT program [43]; inte-
gration and scaling of the reflections were performed with the HKL
Denzo-Scalepack system of programs [44]. Absorption corrections
were carried out using the Gaussian method [45]. The structure
was solved by direct methods with SHELXS-97 [46]. The model was
refined by full-matrix least squares on F2 by means of SHELXL-97
[47]. All hydrogen atoms were stereochemically positioned and

A.C. Roveda Jr. et al. / Inorganica Chimica Acta 409 (2014) 147–155
149
refined with the ridging model except for the ones of the water
molecule which were found from a difference map and refined
with the O–H distance restrained to 0.82(2) Å. Fig. 1 was prepared
using ORTEP-3 for Windows [48]. Listing of atomic coordinates and
equivalent isotropic displacement parameters, full intramolecular
bond distances and angles, hydrogen coordinates, and anisotropic
thermal parameters are available from the authors and were
deposited at the Cambridge Crystallographic Data Centre, reference
numbers CCDC 929821. These data are also shown in Supplemen-
tary material (Tables 1S–3S).
ethanol and dried under vacuum. The obtained products are de-
scribed throughout this text for simplicity as Gx/RuNO (where
x = 0, 2 or 3, and represents PAMAM generations). Ruthenium
nitrosyl attached to PAMAM were hygroscopic, and this property
increased with the increasing of the dendrimers generation.
Yield = 40–45%.
2.7. Nitric oxide release
Nitric oxide release from ruthenium nitrosyl complexes (at-
tached or not to PAMAM) was performed in solution through light
irradiation and by one-electron reduction [12–15].
In the photochemical experiments, samples were irradiated
(kirr = 355 nm) with a Nd:YAG Laser, 10 ns pulse width, attenuated
to approximately 2 mJ/pulse, in a 1.00 cm path length quartz cells
capped with a rubber septum and under constant argon flow. The
progress of the photoreaction, in aqueous solutions pH 2.0
2.6. Functionalization of PAMAM dendrimers with trans-
[Ru(NO)(NH3)4(ina)](BF4)3
Functionalization of PAMAM (G0, G2 and G3) with trans-
[Ru(NO)(NH3)4(ina)](BF4)3 was performed via an amide bond for-
mation using Yamaguchi reagent (2,4,6-trichlorobenzoyl chloride)
[49–51]. Similar procedures were applied for the three PAMAM
generations, as described below.
(
l
= 0.10 mol Lꢂ1 CF3COOH/CF3COONa; CRu ꢁ 9.0 ꢃ 10ꢂ5 mol Lꢂ1),
was monitored spectrophotometrically (UV–Vis). Also, Ru3+, one
of the photoproducts (Eq. (1)) [12,15], was detected through EPR
spectroscopy after the light irradiations (tirr = 30 min) of aqueous
Under absence of air and moisture, a solution containing trans-
[Ru(NO)(NH3)4(ina)](BF4)3 (75 mg, 0.121 mmol) in dry DMF (4 mL)
was treated with triethylamine (Et3N) (0.133 mmol, 20
lL) and
solution of ruthenium nitrosyl complex (CRu ꢁ 2.0 ꢃ 10ꢂ3 mol Lꢂ1
,
2,4,6-trichlorobenzoyl chloride (0.133 mmol, 22 L). The resulting
l
pH 2.0,
l
= 0.10 mol Lꢂ1 CF3COOH/CF3COONa).
suspension was stirred at room temperature (25 °C) for ꢁ24 h
and then, 4-Dimethylaminopyridine (DMAP, 0.242 mmol) and PA-
MAM (0.080 mmol of superficial NH2) dissolved in dry and de-
gassed DMF (ꢁ5 mL) were added in sequence. The mixture was
stirred at room temperature (25 °C) during 96 h for G0, 120 h for
G2 and 168 h for G3. The resulting suspension was filtered and
the solid was washed with DMF and dried under vacuum. The
product was dissolved in ꢁ2 mL of 2.0 mol Lꢂ1 HBF4 solution,
which was filtered and the supernatant was purified by size exclu-
sion chromatography (Sephadex G-25). The first fraction collected
in the chromatographic column was evaporated (rotary evapora-
h
i
3þ
trans- RuIIðNOÞðNH3Þ ðLÞ
4
h
i
ꢃ ꢂ! trans- RuIIIðNH3Þ ðH2OÞðLÞ 3þ þ NO
ð1Þ
hv
4
H2
O
The detection of the nitric oxide released from ruthenium nitro-
syl complexes after light irradiation was performed through EPR
spectroscopy, using the NO spin trap 2-phenyl-4,4,5,5-tetramethy-
limidazoline-1-oxyl-3-oxide (PTIO) [52,53]. Thus, a phosphate buf-
fer solution (pH 7.4, 0.10 mol Lꢂ1 = 0.20 mol Lꢂ1) containing
, l
tor) to
a
minimal volume (ꢁ3–4 mL), and then 1.0 mL of
ruthenium complexes (CRu = 7.5 ꢃ 10ꢂ4 mol Lꢂ1
)
and PTIO
5.0 mol Lꢂ1 HBF4 was added. After the addition of degassed ethanol
(ꢁ15 mL), the solution was cooled in refrigerator and a precipitate
was formed. These solids were collected by filtration, washed with
(CPTIO = 7.5 ꢃ 10ꢂ4 mol Lꢂ1) was irradiated for 5 min. A solution
containing only PTIO was irradiated for 10 min, in the same condi-
tions described above and was used as control.
Fig. 1. ORTEP-3 structure representation for trans-[Ru(NO)(NH3)4(ina)](BF4)(SiF6)ꢀH2O with displacement ellipsoids displayed at 30% probability.

150
A.C. Roveda Jr. et al. / Inorganica Chimica Acta 409 (2014) 147–155
Table 2
For the NO release from chemical reduction, trans-
[Ru(NO)(NH3)4(ina)](BF4)3 (attached or not to PAMAM) was solubi-
Selected bond lengths and bond angles for trans-[Ru(NO)(NH3)4(ina)](BF4)(SiF6)ꢀH2O.
lized in degassed aqueous solution pH 2.0,
l
= 0.10 mol Lꢂ1 (CF3-
Selected bond lengths (Å)
Selected bond angles (°)
COOH/CF3COONa) and then an excess of Eu2+ (4 equivalents) was
added [13,14]. Differential pulse voltammetry (DPV) and electronic
spectroscopy were used to help the detection and characterization
of the produced species.
Ru–N
O–N
1.741(4)
Ru–N–O
N–Ru–N(2)
N–Ru–N(3)
N–Ru–N(4)
176.8(4)
1.132(5)
2.124(3)
2.100(4)
2.116(4)
2.119(4)
2.118(4)
1.323(6)
1.196(6)
94.23(16)
92.04(16)
89.84(16)
93.15(17)
91.74(15)
87.97(15)
92.84(15)
124.0(4)
Ru–N(1)
Ru–N(2)
Ru–N(3)
Ru–N(4)
Ru–N(5)
O(11)–C(1)
O(12)–C(1)
N–Ru–N(5)
N(2)–Ru–N(3)
N(3)–Ru–N(4)
N(5)–Ru–N(4)
O(12)–C(1)–O(11)
3. Results and discussion
3.1. Synthesis
The crystallographic data for trans-[Ru(NO)(NH3)4(ina)](BF4)(-
SiF6)ꢀH2O are listed in the Table 1, and the selected bond lengths
and angles are shown in Table 2. Four NH3 ligands occupy the
equatorial positions and, ina and NO+ ligands are in trans position
in the axial direction (Fig. 1). The mean of the Ru–NH3 distance
is 2.113 0.009 Å and the Ru–NO distance is 1.741(4). These both
values are similar to those found for other nitrosylamminerutheni-
um complexes such as [Ru(NH3)5(NO)]Cl3 [54] and trans-
[Ru(NO)(NH3)4(L)](X)n (where L = N-heterocyclic and X = PF6ꢂ, Clꢂ
or BFꢂ4 ) [12,13]. The Ru–N–O bond angle was 176.8(4)°, which is
compatible with a nitrosonium (NO+) character for the NO ligand
[12]. The ina ligand have two values for the C–O distances in the
carboxyl group, 1.323(6) and 1.196(6) Å, which suggests that one
of the oxygens in this group is protonated, and therefore the ligand
is the isonicotinic acid and not the corresponding anion.
PAMAM dendrimers of generations 0, 2 and 3 have, respec-
tively, 4, 16 and 32 superficial primary amines. Ruthenium com-
plexes were immobilized onto PAMAM via the attachment of
dendrimers terminal amines to the carboxyl group of the complex
trans-[Ru(NO)(NH3)4(ina)](BF4)3 through an amide bond formation
(Fig. 2) [49–51]. Size exclusion chromatography (Sephadex G-25)
was used to isolate the products. Two fractions were separated,
being the first related to the desired product (Gx/RuNO) and the
second, to the excess of trans-[Ru(NO)(NH3)4(ina)](BF4)3.
Two other amidation protocols employing mixed anhydride
(treatment with ethyl chloroformate and triethylamine) [50], and
another using condensation reagents such as DCC/DMAP (Steglich
conditions) failed to give pure products in reasonable yields (less
than 10%).
Another synthetic route was tried out without success through
the attachment of the isonicotinic acid to dendrimers [49–51]
(Fig. 1S, Supplementary material). Then, this product was submit-
ted to the reaction with trans-[Ru(NH3)4(SO2)(Cl)]Cl, following the
sulfite/sulfate route [12–14]. The problem with this route was the
oxidation step of the metal center (Ru2+ to Ru3+) and of the ligand
(SO2 to SO2ꢂ) using hydrogen peroxide [12–14,55]. This oxidation
step needs to be carried out before the nitric oxide coordination
to ruthenium, but it unfortunately compromised the dendrimer
structure.
4
3.2. Spectroscopy
The nitrosyl complexes (attached or not to PAMAM) are EPR si-
lent, indicating that no Ru3+ is formed on the anchoring of the com-
plex to dendrimers.
As expected, the 1H NMR spectra of Gx/RuNO (x = 0, 2 and 3)
showed similarities to the 1H NMR spectra of the synthesis precur-
sors: Fig. 3 for G0/RuNO, and Figs. 2S and 3S (Supplementary mate-
rial) for G2/RuNO and G3/RuNO, respectively. As can be noticed
comparing the 1H NMR spectra of PAMAM G0 and of G0/RuNO in
Fig. 3, the signals with chemical shift (d) between 2.7 and
4.0 ppm are related to the dendrimer’s moiety of the G0/RuNO
product. This is an indication of the carboxyl attachment (of the
ina ligand) to the terminal PAMAM amines. The only significant
differences between the 1H NMR spectra of PAMAM (G0, G2 and
G3) and Gx/RuNO (x = 0, 2 and 3) is the presence of two doublets,
centered at d = 8.36 and 8.75 ppm for the Gx/RuNO species. These
signals are also presented in the 1H NMR spectrum of trans-
[Ru(NO)(NH3)4(ina)]3+, with d = 8.36 and 8.75 ppm (Fig. 4S, Supple-
mentary material), and their presence is consistent with the coor-
dination of the N-heterocyclic ligand to the metal center through
the pyridine-type nitrogen [13,14].
An attempt to estimate the number of PAMAM’s superficial
amines functionalized with the ruthenium complex was carried
out through the integration of the hydrogen signals in the 1H
NMR spectra (Fig. 3 and Figs. 2S and 3S; Tables 4S and 5S, Supple-
mentary material). The integrations were performed using the area
of the characteristic aromatic hydrogens peaks of the ina ligand
(which corresponds to four H for each ina ligand, Fig. 2) and the
area of the aliphatic hydrogens of the PAMAM dendrimers
(Fig. 2). These results are summarized in Table 3.
Table 1
Crystallographic data for trans-[Ru(NO)(NH3)4(ina)](BF4)(SiF6)ꢀH2O.
An alternative method to estimate the number of ruthenium
nitrosyl attached to PAMAM was performed by analyzing the
ruthenium content through ICP-OES (Table 3). The ratio between
the theoretical (Gx fully functionalized) and the experimental con-
centration of ruthenium for a known mass of Gx/RuNO was used as
an indication of the degree of the trans-[Ru(NO)(NH3)4(ina)](BF4)3
incorporation on the dendrimer.
Formula
C6H19BF10N6O4RuSi
Crystal system
Space group
a (Å)
b (Å)
c (Å)
monoclinic
P21/n
7.9616(2)
24.3982(8)
9.9705(3)
90
98.781(2)
90
1914.06(10)
4
0.71073
1.975
0.0473
0.1153
0.0626
0.1246
a
(°)
b (°)
According to the 1H NMR and ICP-OES data, PAMAM G0 was
fully functionalized with trans-[Ru(NO)(NH3)4(ina)](BF4)3, and G2
and G3 were functionalized with ꢁ8 and ꢁ27 ruthenium nitrosyls,
respectively. Thus, according to these results, the compounds G0/
RuNO, G2/RuNO and G3/RuNO described in this work were able
c
(°)
V (Å3)
Z
Wavelength (Å)
qcalc (Mg m3)
R1 [I > 2
r
(I)]
wR2
R1 (all data)
wR2 (all data)
to storage ꢁ1.43, ꢁ1.03 and ꢁ1.28
lmol NO per mg of compound,
respectively. Analogous results were obtained by Benini and
collaborators [30], in which PAMAM G0, G2 and G3 were

A.C. Roveda Jr. et al. / Inorganica Chimica Acta 409 (2014) 147–155
151
Fig. 2. Functionalization of PAMAM G0 with trans-[Ru(NO)(NH3)4(ina)](BF4)3.
functionalized, respectively, with ꢁ4, ꢁ12 and ꢁ29 [RuIII(edta)(H2-
O)]ꢂ complexes.
observed for the free complex [30], the number of functionalized
amines was estimated and these results are shown in Table 6S (Sup-
plementary material). Using the same approach was possible to cal-
culate ꢁ8 and ꢁ27 trans-[Ru(NO)(NH3)4(ina)](BF4)3 attached to
PAMAM G2 and G3, respectively. These results are consistent with
the ones calculated from NMR and ICP-OES data (Table 3). Never-
theless, the approach using only UV–Vis is not conclusive and must
be performed together with other techniques (as 1H NMR and
ruthenium analysis) in order to obtain a more reliable value.
The formal reduction potentials of the RuIINO+/RuIINO0 couple
The electronic spectrum of trans-[Ru(NO)(NH3)4(ina)]3+ showed
intense bands at 232 nm (
e
= 11.2 0.3 ꢃ 103 Mꢂ1 cmꢂ1) and at
275 nm (
e
= 3.9 0.2 ꢃ 103 Mꢂ1 cmꢂ1), assigned as internal ligand
transitions (IL) of ina; one band at 335 nm (e
= 221.9 7.3 Mꢂ1
cmꢂ1), assigned to Ru d–d transitions, with contributions from a
MLCT transition (d weaker band at
Ru(II) ? p⁄(NO)), and
466 nm (
= 23.9 6.9 Mꢂ1 cmꢂ1) assigned to d Ru(II) ? p⁄(NO)
and
(ina) ? p⁄(NO) transitions. These above assignments were
p
a
e
p
p
tentatively carried out by analogy to previously reported data for
other ruthenium tetraammine nitrosyls [12–14]. The UV–Vis spec-
tra of Gx/RuNO (x = 0, 2 and 3) exhibited the absorption maxima
governed by the ruthenium nitrosyl moiety (Fig. 5S, Supplemen-
tary material). These functionalized dendrimers (Gx/RuNO)
showed bands at 232, 275, 335 and 466 nm, and the assignments
for these bands were similar to the ones described above for
trans-[Ru(NO)(NH3)4(ina)]3+. PAMAM G0, G2 and G3 have a band
ðENOþ
Þ for trans-[Ru(NO)(NH3)4(ina)]3+ and for Gx/RuNO (x = 0,
=NO0
2 and 3) in solution, were similar, ꢁ60 mV versus NHE (Table 4).
Analogous results were obtained for other ruthenium nitrosyl
immobilized on PAMAM dendrimers [30] and in other matrices
[17]. It is interesting to recall that these potentials are in the acces-
sible range for biological reducing agents [7,57], which potentially
enables the title compounds as candidates for therapy use.
The m
(NO+) frequencies for the complex trans-[Ru(NO)(NH3)4(-
ina)](BF4)3 (attached or not to PAMAM) are also presented in Ta-
ble 4. All the ruthenium compounds described in this work
at kmax = 278 nm (
e
= 130–150 Mꢂ1 cmꢂ1). This low intensity band,
in comparison to that observed for trans-[Ru(NO)(NH3)4(ina)]3+ in
the same region (275 nm,
e
= 3.9 0.2 ꢃ 103 Mꢂ1 cmꢂ1), confirms
showed
a
medium to strong intensity band in the 1930–
(NO+) (Figs. 6S–8S, Supplementary
the small contribution of the dendrimer moiety for the absorption
maxima in the UV–Vis spectra of Gx/RuNO products. Similar
results were found by Stasko and collaborators [29], which
functionalized two PAMAM G4 dendrimers with S-nitrosothiol
1933 cmꢂ1 range ascribed to
m
material), which is compatible with a nitrosonium (NO+) character
of the NO ligand (as previously discussed in the crystallographic
results in the Section 3.1) [12]. Comparing the FT-IR spectra of
the products (Gx/RuNO) with the starting compounds is also pos-
sible to notice the appearance of a medium intensity band in the
region of 1640–1680 cmꢂ1 for Gx/RuNO (x = 0, 2 and 3), assigned
(N-acetyl-D,L-penicillamine or N-acetyl-
L-cysteine), and also by
Benini et al. [30].
According to the discussed above, using the
e
values for k = 232
and 275 nm for trans-[Ru(NO)(NH3)4(ina)](BF4)3, would be possible
to the amide I (mCO) [30,42,58], which is present on PAMAM (G0,
to estimate the number of the PAMAM amines functionalized with
G2 and G3) but is absent in the complex trans-[Ru(NO)(NH3)4(-
ina)](BF4)3 (Figs. 6S–8S, Supplementary material). This is an indic-
ative of the amide bond formed between the ina ligand and the
dendrimer. Other differences observed in the FT-IR spectra of Gx/
the ruthenium nitrosyl complex [30,56]. Thus, assuming that the
e
values for the G0 functionalized with four trans-[Ru(NO)(NH3)4(-
ina)](BF4)3, would be in principle approximately four times of that

152
A.C. Roveda Jr. et al. / Inorganica Chimica Acta 409 (2014) 147–155
Fig. 3. 1H NMR spectra of (A) PAMAM G0 and (B) G0/RuNO (1.0 mol Lꢂ1 CF3COOD in D2O).
Table 3
Estimative of the PAMAM’s superficial amines functionalized with trans-[Ru(NO)(NH3)4(ina)](BF4)3 performed by 1H NMR and ICP-OES.
Compounds
Number of PAMAM’s superficial aminesa
Number of functionalized amines
1H NMR
ICP-OESb
G0/RuNO
G2/RuNO
G3/RuNO
4
16
32
4
7.7
28.1
3.7 0.21
7.3 0.65
26.3 0.83
a
Superficial amines of PAMAM G0, G2 and G3
Results from three independent assays.
b
RuNO in relation to that of trans-[Ru(NO)(NH3)4(ina)](BF4)3 are the
dendrimers with trans-[Ru(NO)(NH3)4(ina)](BF4)3 did not signifi-
cantly alter the spectroscopic and electrochemical characteristics
of the ruthenium complex.
appearance of strong broad bands around 3070–3080 cmꢂ1
(m (m(NH) and m(CH)) [42] for Gx/RuNO,
(NH)) and 2940–2960 cmꢂ1
which are present in PAMAM (G0, G2 and G3) dendrimers [58] in
the same region (Figs. 6S–8S, Supplementary material) and are ab-
sent in the FT-IR spectrum of trans-[Ru(NO)(NH3)4(ina)](BF4)3.
Thus, the electrochemical (DPV and CV) and spectroscopic
(UV–Vis and FT-IR) results suggest that the functionalization of
3.3. Photochemical NO release
NO release from ruthenium tetraammine nitrosyl (Eq. (1)) can
be achieved by irradiation of these complexes in solution with light

A.C. Roveda Jr. et al. / Inorganica Chimica Acta 409 (2014) 147–155
153
Table 4
Electrochemical data and
m(NO) frequencies for ruthenium nitrosyl complexes
attached or not to PAMAM dendrimer.
b
Compound
EcpðNOþ
(mV vs. NHE)a
Þ
m )
(NO+)(cmꢂ1
=NO0
trans-
59
1933
[Ru(NO)(NH3)4(ina)](BF4)3
G0/RuNO
G2/RuNO
G3/RuNO
57
58
59
1931
1930
1931
a
Potential of the cathodic peak obtained by differential pulse voltammetry, pH
= 0.10 M (CF3COOH/CF3COONa), T = (25 1) °C; scan rate = 5 mV/s, pulse
2.0,
l
height = 50 mV.
b
KBr pellet, resolution of 4 cmꢂ1
.
in the range of 300–370 nm [15]. Thus, solutions containing trans-
[Ru(NO)(NH3)4(ina)]3+ (attached or not to PAMAM G2) were irradi-
ated at 355 nm in phosphate buffer (pH 7.4) in the presence of
PTIO. This last compound is a well known nitric oxide spin trap
[52,53]. PTIO reacts with NO yielding PTI, as illustrated in Fig. 4.
Both PTIO and PTI are paramagnetic species, and therefore can be
detected and distinguished through EPR spectroscopy [52,53].
Thus, performing the photochemical reaction for trans-
[Ru(NO)(NH3)4(ina)]3+ and for G2/RuNO in the presence of PTIO,
it was possible to detect the NO release from these compounds
due to the observation of the signals related to the PTI species
(Fig. 5) [52,53]. The values obtained for the hyperfine splitting con-
stant (Fig. 5) were: aN1,3 = 0.82 mT for PTIO; aN1 = 0.98 mT and
aN3 = 0.44 mT for PTI, which are consistent with the ones reported
previously [52].
Fig. 5. EPR detection of NO release from the ruthenium nitrosyl upon photochemical
reaction. EPR spectra of irradiated solutions containing: (A) only PTIO (control),
irradiated for 10 min; (B) PTIO + trans-[Ru(NO)(NH3)4(ina)]3+ and (C) PTIO + G2/
RuNO, both irradiated for 5 min. (phosphate buffer solutions pH 7.4; CRu = 7.5 -
ꢃ 10ꢂ4 mol Lꢂ1, CPTIO = 7.5 ꢃ 10ꢂ4 mol Lꢂ1; kirr = 355 nm, ꢁ2 mJ/pulse; T = 25 1 °C).
to achieve a more accessible irradiation region to be used in pho-
todynamic therapy.
3.4. NO release from chemical reduction
This photochemical reaction was also performed for trans-
[Ru(NO)(NH3)4(ina)]3+ and for G3/RuNO, in the absence of PTIO,
It is well established that the NO dissociation from trans-
[Ru(NO)(NH3)4(L)]3+ complexes can be triggered by the reaction
with Eu2+ ions [12–14], as shown in Eqs. (2) and (3).
in aqueous solution (pH 2.0,
l
= 0.10 mol Lꢂ1 CF3COOH/CF3-
COONa), and it was monitored through UV–Vis spectroscopy
(Fig. 9S, Supplementary material). It was possible to observe an
absorbance decrease at ꢁ230 nm concomitant with an absorbance
increase at 275 nm and at 300–350 nm. Also an isosbestic point
was observed at 249 nm. This photochemical behavior for the
nitrosyl complex (attached or not to PAMAM) was similar to that
previously described for other ruthenium tetraammine nitrosyls
in solution [15] or immobilized in different matrices [17,22]. The
formation of the trans-[RuIII(NH3)4(H2O)(ina)]3+ photoproduct
h
i
3þ
trans- RuIIðNOþÞðNH3Þ ðLÞ
4
h
i
þ Eu2þꢂ!trans- RuIIðNO0ÞðNH3Þ ðLÞ 2þ þ Eu3þ
ð2Þ
4
h
i
2þ
trans- RuIIðNO0ÞðNH3Þ ðLÞ
4
h
i
þ H2O ꢂ! trans- RuIIðNH3Þ ðH2OÞðLÞ 2þ þ NO
ð3Þ
k
ꢂNO
[15] (pH 2.0,
l
= 0.10 mol Lꢂ1, CF3COOH/CF3COONa) was confirmed
4
through EPR spectroscopy (Fig. 6) [12,14,15].
The g-factor for trans-[RuIII(NH3)4(H2O)(ina)]3+ (not attached to
PAMAM) is g = 2.543, and for trans-[RuIII(NH3)4(H2O)(ina)]3+ at-
tached to PAMAM G3 are g\ = 2.481 and g|| = 2.675. The other g-
factor for trans-[RuIII(NH3)4(H2O)(ina)]3+ (not attached to PAMAM)
was not observed and this difficulty was previously reported and
thoroughly discussed elsewhere [55,59]. Also, when comparing
the EPR spectra for trans-[RuIII(NH3)4(H2O)(ina)]3+ attached or not
to PAMAM (Fig. 6), a shoulder can be observed in the G3/RuNO
spectrum. This was tentatively attributed to matrix effects [17].
Furthermore, as far as the compounds described in this work
are concerned, the photochemical NO release activation pathway
requires light excitation in a non favorable region of the spectra
(k 6 400 nm). Thus some coordination sphere tailoring is necessary
Thus, the NO dissociation from ruthenium nitrosyl attached to
PAMAM was similarly investigated through the reaction with
Eu2+ ions. As observed in Fig. 7, the DPV of a solution containing
G3/RuNO showed a cathodic peak (Ecp1) at ꢁ0.060 V versus NHE,
ascribed to the RuNO+/RuNO0 process. After the reaction of G3/
RuNO with Eu2+, the process described above disappeared, whereas
a new one was observed with Eap1 ꢁ 0.295 V versus NHE, which
was assigned to the [RuH2O]3+/[RuH2O]2+ couple in trans-
[Ru(NH3)4(H2O)(ina)]2+ (Fig. 7). Also, an electrochemical anodic
wave with Eap2 ꢁ 0.753 V versus NHE can be observed. This last
electrochemical process is due to the oxidation of free NO in solu-
tion [14,60], confirming that the NO was released from G3/RuNO
after the reaction with Eu2+
.
The reaction between Gx/RuNO and Eu2+ was also investigated
spectrophotometrically, as shown in the Fig. 8 for G3/RuNO. After
the addition of Eu2+ to the solution containing G3/RuNO, a new
band at 492 nm appeared, consistent with the trans-[Ru(NH3)4
(H2O)(ina)]2+ formation, and which was assigned to a metal to
ligand charge transfer (MLCT) dp
Ru(II) ? p⁄(ina) [12–14].
Therefore, as suggested by DPV and electronic spectroscopy
data, the ions trans-[Ru(NO)(NH3)4(ina)]3+ attached to PAMAM,
after reduction, exhibited nitric oxide dissociation and the respec-
Fig. 4. PTIO conversion into PTI through the reaction with NO [53, 54].
tive formation of the aqua species trans-[Ru(NH3)4(H2O)(ina)]2+
.

154
A.C. Roveda Jr. et al. / Inorganica Chimica Acta 409 (2014) 147–155
Fig. 6. EPR spectra of solutions containing (A) trans-[Ru(NO)(NH3)4(ina)]3+ and (B) G3/RuNO after 30 min of laser irradiation (CRu ꢁ 2.0 ꢃ 10ꢂ3 mol Lꢂ1
; pH 2.0,
l
= 0.10 mol Lꢂ1 CF3COOH/CF3COONa; kirr = 355 nm, ꢁ2 mJ/pulse; T = 25 1 °C. EPR spectra were recorded at T = 77 K).
Fig. 8. Spectral changes during the reaction between G3/RuNO and Eu2+. Initial
spectrum of G3/RuNO (dashed line). The first spectrum was recorded 30s after the
addition of Eu2+ to the solution containing G3/RuNO, and the next ones, every 1 min
Fig. 7. Differential pulse voltammograms of the reaction between G3/RuNO and
Eu2+. DPV of G3/RuNO before (black line) and after (gray line) the reaction with
Eu2+. (CRu = 5.0 ꢃ 10ꢂ4 mol Lꢂ1; CEu2+ = 2.0 ꢃ 10ꢂ3 mol Lꢂ1; pH 2.0,
l
= 0.10 mol Lꢂ1
(CRu = 6.5 ꢃ 10ꢂ5 mol Lꢂ1; CEu2+ = 2.6 ꢃ 10ꢂ4 mol Lꢂ1; pH 2.0,
COOH/CF3COONa; T = 25 1 °C).
l
= 0.10 mol Lꢂ1, CF3-
CF3COOH/CF3COONa; scan rate = 5 mV/s; pulse height = 50 mV; T = 25 1 °C).
Toledo et al. [57] found a correlation between the rate constant
S-nitrosothiols [29] release NO by two main pathways: photo-ini-
tiated decomposition, with t1/2 in the range of 34–200 min, and
transition metal-mediated catalytic decomposition (based on
Cu+/Cu2+ redox couple) with t1/2 in the range of 1.5–106 min
[29]. PAMAM functionalized with [Ru(edta)(NO)]ꢂ [30] release
NO in an analogous way to that observed for the compounds de-
scribed in this work (by chemical reduction) with t1/2 in the range
of 4–8 min.
Since the NO liberation from ruthenium nitrosyl compounds
can be triggered on a controlled way by photochemical and ther-
mal activation (reduction), these compounds offer the possibility
of designing and exploring them as new platforms for NO
transport.
for NO dissociation (kꢂNO), represented in Eq. (3), and the sum of
P
the electrochemical parameters ( EL), introduced by Lever [61],
as shown in Eq. (4):
X
K
¼ ðꢂ0:81 ꢃ
ELÞ þ 0:48; ðwith R ¼ 0:997Þ
ð4Þ
ꢂNO
P
Using this correlation (with EL = 0.57) [57,61], it was possible
to estimate the rate constant for the NO dissociation from trans-
[Ru(NO)(NH3)4(ina)]2+ as kꢂNO ꢁ 0.0183 sꢂ1, with t1/2 ꢁ 38 s. Since
no drastic changes were observed between the ruthenium nitrosyl
properties in solution or attached to dendrimers [30] it is likely
that the k
for Gx/RuNO would be around 0.02 sꢂ1 [30].
ꢂNO
The NO release mechanism for the NO-releasing dendrimers
described in the literature [18–20,29,30,62] is diversified. Dendri-
mers functionalized with N-diazeniumdiolates [18–20] release NO
spontaneously in physiological conditions with a t1/2 for NO re-
lease varying from 1.4–293 min, depending on the dendrimers
surface modifications[18,20,62]. Dendrimers functionalized with
Therefore, when dealing with hypoxic conditions, as in the
infections like Chaga’s Disease and Leishmaniosis, or even in solid
tumors, the environment might provide the necessary reducers for
the RuIINO+/RuIINO0 reduction in the nitrosyl compounds. Thus,
Gx/RuNO could be good candidates to be used in these therapies.

A.C. Roveda Jr. et al. / Inorganica Chimica Acta 409 (2014) 147–155
155
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ried out with the compounds Gx/RuNO against Trypanosoma cruzi
(Y strain). Results collected to date indicate that the trypanocidal
activity of G0/RuNO and G2/RuNO (CRu = 200 ꢃ 10ꢂ6 mol Lꢂ1) were
approximately 82% and 88%, respectively. These compounds exhib-
ited a trypanocidal activity slightly higher than that observed for
the complex trans-[Ru(NO)(NH3)4(ina)](BF4)3 (72% of trypanocidal
activity for CRu = 200 ꢃ 10ꢂ6 mol Lꢂ1). The in vivo experiments, in
which these differences might be more evident, are in course and
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