Reactivity of Radicals Generated on Irradiation of trans-[Ru(NH 3)4(NO2)P(OEt)3](PF6)

Article abstract of DOI:10.1021/ja0373263

The electronic absorption spectrum of trans-[Ru(NH₃) ₄(NO₂)(P(OEt)₃]⁺ in aqueous solution is characterized by a strong absorption band at 334 nm (λ _max = 1800 mol^-1 L cm^-1). On the basis of quantum mechanics calculations, this band has been assigned to a MLCT transition from the metal to the nitro ligand. Molecular orbital calculations also predict an LF transition at 406 nm, which is obscured by the intense MLCT transition. When trans-[Ru(NH₃)₄(NO ₂)(P(OEt)₃]⁺ in acetonitrile is irradiated with a 355 nm pulsed laser light, the absorption features are gradually shifted to represent those of the solventocomplex trans-[Ru(NH₃) ₄(solv)(P(OEt)₃]²⁺ (λ_max = 316 nm, ε = 650 mol^-1 L cm^-1), which was also detected by ³¹P NMR spectroscopy. The net photoreaction under these conditions is a photoaquation of trans-[Ru(NH₃)₄-(NO ₂)(P(OEt)₃]⁺, although, after photolysis, the presence of the nitric oxide was detected by differential pulse polarography. In phosphate buffer pH 9.0, after 15 min of photolysis, a thermal reaction resulted in the formation of a hydroxyl radical and a small amount of a paramagnetic species as detected by EPR spectroscopy. In the presence of trans-[Ru(NH₃)₄(solv)P(OEt)₃]²⁺, the hydroxyl radical initiated a chain reaction. On the basis of spectroscopic and electrochemical data, the role of the radicals produced is analyzed and a reaction sequence consistent with the experimental results is proposed. The 355 nm laser photolysis of trans-[Ru(NH₃)₄(NO ₂)(P(OEt)₃]⁺ in phosphate buffer pH 7.4 also gives nitric oxide, which is readily trapped by ferrihemeproteins (myoglobin, hemoglobin, and cytochrome C), giving rise to the formation of their nitrosylhemeproteins(II), (NO)Fe(II)hem.

Full text of DOI:10.1021/ja0373263

Published on Web 02/06/2004
Reactivity of Radicals Generated on Irradiation of
trans-[Ru(NH3)4(NO2)P(OEt)3](PF6)
Rose Maria Carlos,† Daniel Rodrigues Cardoso,† Eduardo Ernesto Castellano,‡
Renata Zachi Osti,† Ademir Joa˜o Camargo,† Luis Guilherme Macedo,† and
Douglas Wagner Franco*,‡
Contribution from the Instituto de Qu´ımica de Sa˜o Carlos, IQSC-USP, AVenida Trabalhador
Sa˜oCarlense, 400, CP 780, CEP 13560-970, Sa˜o Carlos - SP, Brazil, and Instituto de F´ısica de
Sa˜o Carlos, IFSC-USP, AVenida Trabalhador Sa˜oCarlense, 400, CP 369, CEP13566-590,
Sa˜o Carlos - SP, Brazil
Received July 16, 2003; E-mail: douglas@iqsc.usp.br
Abstract: The electronic absorption spectrum of trans-[Ru(NH3)4(NO2)(P(OEt)3]+ in aqueous solution is
characterized by a strong absorption band at 334 nm (λmax ) 1800 mol-1 L cm-1). On the basis of quantum
mechanics calculations, this band has been assigned to a MLCT transition from the metal to the nitro
ligand. Molecular orbital calculations also predict an LF transition at 406 nm, which is obscured by the
intense MLCT transition. When trans-[Ru(NH3)4(NO2)(P(OEt)3]+ in acetonitrile is irradiated with a 355 nm
pulsed laser light, the absorption features are gradually shifted to represent those of the solventocomplex
trans-[Ru(NH3)4(solv)(P(OEt)3]2+ (λmax ) 316 nm, ꢀ ) 650 mol-1 L cm-1), which was also detected by 31P
NMR spectroscopy. The net photoreaction under these conditions is a photoaquation of trans-[Ru(NH3)4-
(NO2)(P(OEt)3]+, although, after photolysis, the presence of the nitric oxide was detected by differential
pulse polarography. In phosphate buffer pH 9.0, after 15 min of photolysis, a thermal reaction resulted in
the formation of a hydroxyl radical and a small amount of a paramagnetic species as detected by EPR
spectroscopy. In the presence of trans-[Ru(NH3)4(solv)P(OEt)3]2+, the hydroxyl radical initiated a chain
reaction. On the basis of spectroscopic and electrochemical data, the role of the radicals produced is
analyzed and a reaction sequence consistent with the experimental results is proposed. The 355 nm laser
photolysis of trans-[Ru(NH3)4(NO2)(P(OEt)3]+ in phosphate buffer pH 7.4 also gives nitric oxide, which is
readily trapped by ferrihemeproteins (myoglobin, hemoglobin, and cytochrome C), giving rise to the formation
of their nitrosylhemeproteins(II), (NO)Fe(II)hem.
Introduction
laboratory is interested in studying ruthenium(II) ammine
complexes that may serve as potential photochemical precursors
Nitric oxide is widely recognized as a biologically important
molecule, regulating blood pressure, acting as a neurotransmitter,
and participating in the ability of the immune system to kill
tumor cells and intracellular parasites.1-5 Regarding the pho-
tochemical behavior of nitric oxide, coordination complexes of
iron, cobalt, chromium, and ruthenium with nitrosyl groups have
been investigated to develop molecular devices for the delivery
of nitric oxide and targeting processes.6-9 In this respect, our
of nitric oxide.10-15 Previous studies have demonstrated the pho-
tolability of trans-[Ru(NH3)4(NO)L]3+ complexes thereby re-
leasing nitric oxide in aqueous solution.15 These complexes
exhibit desirable qualities such as water solubility, stability, and
toxicity10-12,15 for the delivery of nitric oxide to biological tar-
gets, while quantum yields are elevated (0.1-0.3 mol einstein-1).
However, their absorptivities are relatively low (200-600 mol-1
L cm-1). To increase the scope of ruthenium complexes, trans-
[Ru(NH3)4(NO2)P(OEt)3]+ has been synthesized, for which the
ligand field is weaker than that for the nitrosyl containing the
complex. Furthermore, the trans-[Ru(NH3)4(NO2)P(OEt)3]+
complex exhibits an intense charge-transfer transition (ꢀ ≈ 1800
† IQSC-USP.
‡ IFSC-USP.
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S. C.; Franco, D. W. Inorg. Chem. 1998, 37, 2670-2677.
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S.; Franco, D. W. J. Chem. Soc., Dalton Trans. 1998, 601-607.
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Acta 2000, 300, 698-708.
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2002, 41, 3728-3739.
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ReV. 2003, 236, 57-69.
9
2546
J. AM. CHEM. SOC. 2004, 126, 2546-2555
10.1021/ja0373263 CCC: $27.50 © 2004 American Chemical Society

Radicals of trans-[Ru(NH ) (NO2)P(OEt)3](PF6)
A R T I C L E S
3 4
Figure 1. A view from the DFT calculation of the coordinated geometry of trans-[Ru(NH3)4(NO2)P(OEt)3]+. Inset: Orientation of the axis adopted by the
DFT calculation.
mol-1 L cm-1) in the 330 nm region. The present paper de-
in 40 mL of degassed CF3COOH (pH 2.0). Exhaustive electrolysis at
+0.70 V vs SCE using the Pt plate as working and counter electrodes
and an Ag wire coated by AgCl in a capillary as the reference electrode
provided trans-[Ru(NH3)4(H2O)P(OEt)3]3+. When the oxidation was
complete, the solvent was removed, and the residue was washed with
ethanol and ether.
scribes the X-ray data, molecular orbital calculations, UV-vis
spectral data, and photochemical properties of trans-[Ru(NH3)4-
(NO2)P(OEt)3]+ in both aqueous phosphate buffer and aceto-
nitrile. The thermal and photochemical reactions of trans-
[Ru(NH3)4(NO2)(P(OEt)3]+, in phosphate pH 7.4 containing
hemeproteins (myoglobin(III), hemoglobin(III), and cytochrome
C), are also presented.
Crystal Data and Data Collection. Crystals of C6H27F6N5O5P2Ru
were obtained from an aqueous solution maintained in the refrigerator
for 4 days, under argon atmosphere. A yellow crystal fragment (0.10
mm × 0.12 mm × 0.12 mm) of the complex was used in the single-
crystal X-ray diffraction experiment. All measurements were taken in
an Enraf-Nonius Kappa CCD diffractometer with graphite monochro-
mated Mo KR (λ ) 0.71073 Å) radiation. C6H27F6N5O5P2Ru, FW )
526.34, T ) 120(2) K, λ ) 0.71073 Å, orthorhombic, space group
P212121, a ) 10.134(1) Å, b ) 10.200(1) Å, c ) 19.395(1) Å, V )
2004.80(8) Å3, Z ) 4, F ) 1.744 Mg (m3)-1, µ ) 1.021 mm-1, F(000)
) 1064. Data collection range 2.91-26.0°, -12 e h e 11, -12 e k
e 12, -23 e l e 23. Reflections collected/unique 11 811/3860 (R(int)
) 0.049).
Experimental Section
Materials. Analytical-grade chemicals and HPLC grade solvents
were used for all experiments. Acetonitrile was stored over molecular
sieves. Oxidized forms of hemoglobin (human), myoglobin (horse
heart), and cytochrome C (horse heart) were obtained from Sigma and
used as supplied. The pH values (pH 7-8) of phosphate buffer (ionic
strength, 0.2) were adjusted with the use of aqueous solutions of 0.025
mol L-1 KH2PO4 and 0.025 mol L-1 K2HPO4. At a pH higher than
8.0, 0.05 mol L-1 Na2BO4 buffer was used.
trans-[Ru(NH3)4(H2O)P(OEt)3](PF6)2 was obtained as a white solid
following the literature procedure.16,17 trans-[Ru(NH3)4(NO2)P(OEt)3]-
(PF6) was synthesized by the reaction of 200 mg of trans-[Ru(NH3)4-
(H2O)P(OEt)3]2+ and 250 mg of NaNO2 in 2 mL of degassed Milli-Q
water at room temperature. After 30 min of reaction, 200 mg of
NH4PF6 was added, and the yellow solution was kept in the refrigerator
for 12 h. trans-[Ru(NH3)4(NO2)P(OEt)3](PF6) was collected by filtration,
washed with diethyl ether, and dried under vacuum. The yield was ca.
65%. The solid complex was stable for several days when stored in
the dark under inert atmosphere. Anal. Calcd: Ru, 19.2; C, 13.7; N,
13.3; H, 5.2. Found: Ru, 18.5; C, 14.0; N, 13.9; H, 5.3. IR data: 1633
cm-1 (w), δ NH3; 1400 cm-1 (s), νa NO2; 1024 cm-1 (s), δ P-O-C;
834 cm-1 (s) ν PF6; and 560 cm-1 (s) δ PF6.
Structure Solution and Refinement. The structure was solved by
direct methods with SHELXS-97.19 The Fourier map obtained showed
all non-hydrogen atoms. The model was refined by full-matrix least-
squares procedure on F 2 by means of SHELXL-97.19 The hydrogen
atoms of the CH2 groups were set isotropic with a thermal parameter
20% greater than the equivalent isotropic displacement parameter of
the carbon atom to which each one is bonded; this percentage was set
to 50% for the hydrogen atoms of the NH3 and CH3 groups. All of the
hydrogen atoms were stereochemically positioned and refined with the
riding model.19 Final R indices [3771 reflections with I > 2σ(I)] ) R1
) 0.0246, wR2 ) 0.0606, R indices (all data) R1 ) 0.0256, wR2 )
0.0612. Absolute structure parameter -0.01 (2). Largest difference peak
and hole 0.357 and -0.656 e Å-3. An ORTEP20,21 projection of the
complex is shown in Figure 1.
trans-[Ru(NH3)4(H2O)P(OEt)3](PF6)3 was generated by electrolysis
of the corresponding Ru(II) complex.18 In a typical experiment, a sample
of ca. 200 mg of trans-[Ru(NH3)4(H2O)P(OEt)3](PF6)2 was dissolved
(18) Mazzeto, S. E.; Rodrigues, E.; Franco, D. W. Polyhedron 1993, 12, 971-
975.
(16) Franco, D. W.; Taube, H. Inorg. Chem. 1978, 17, 571-578.
(17) Rezende, N. M. S.; Martins, S. C.; Marinho, L. A.; Santos, J. A. V.; Tabak,
M.; Perussi, J. R.; Franco, D. W. Inorg. Chim. Acta 1991, 182, 87-92.
(19) Sheldrick, G. M. SHELX97, Programs for Crystal Structure Analysis
(Release 97-2); Institu¨t fu¨r Anorganische Chemie der Universita¨t: Tam-
manstrasse 4, D-3400 Go¨ttingen, Germany, 1998.
9
J. AM. CHEM. SOC. VOL. 126, NO. 8, 2004 2547

A R T I C L E S
Carlos et al.
Table 1. Selected Bond Lengths (Å) and Angles (deg)
Electrochemical NO Detection. The release of NO in CH3CN was
monitored using an EG&G Princeton Applied Research model 264A
polarographic analyzer/stripping voltammeter in conjunction with a three
electrode cell. Differential pulse voltammograms were obtained in
CH3CN at 22 °C in a voltammetric cell with a glassy-carbon electrode
as the working electrode, a platinum wire as the auxiliary, and an SCE
reference electrode. Nitric oxide quantitative measurements were per-
formed using an NO sensor electrode from Innovative Instruments, Inc.,
a model amiNO700 electrode with an inNO Nitric Oxide Measuring
System.
Photochemistry. The complex was irradiated using the third
harmonic (355 nm) of an Nd:YAG 10 ns pulse width laser (Continuum
Surelite OPO IIP-10) attenuated to approximately 2 mJ/pulse as the
excitation source. The progress of the photoreaction was monitored
spectrophotometrically in a Hitachi model U2000 spectrophotometer.
The photolysis was performed at 25 °C in 1.0 cm path length quartz
cells capped with a rubber septum. The solutions (10-3-10-4 mol L-1
initial complex concentration) were deaerated by bubbling with argon
prior to photolysis and were stirred during irradiation. The solutions
were photolyzed to approximately 10% conversion. Dark reactions were
performed simultaneously using solutions of identical composition. As
analyzed from their UV-vis spectra, these samples were stable in the
dark in a time scale longer than that of the photochemical experiments.
Photoproducts were identified by comparing their UV-vis spectra with
the spectra of solvent-substituted ruthenium(II) phosphite complex16,17
trans-[Ru(NH3)4(H2O)P(OEt)3]2+. Compounds formed by dark reactions
were identified by NMR, EPR, and electrochemical (differential pulse
voltammetry and electrolysis) techniques.
31P NMR spectra were obtained using 5 mm vacuum NMR tubes
(Wilmad) in a Bruker DRX 400 9.4 T (161.9 MHz to 31P) spectrometer.
The sample (ca. 30 mg) was dissolved in the appropriate solvent, and
D2O served as a reference. The dearation of solutions was achieved
with 3-5 vacuum-argon cycles in a Schlenk line.
EPR measurements were taken using a quartz flat cell at room
temperature or 2 mm quartz tubes for 77 K (Wilmad) in a Bruker ESP
300E X-band spectrometer equipped with a TE102 standard cavity.
The experiment at 77 K was performed using a quartz Dewar filled
with liquid nitrogen.
Heme Proteins Nitrosylation. All of the sample solutions were
prepared in the vacuum line with meticulous care to avoid oxygen
contamination. A glass apparatus used for the present study consists
of a round-bottomed vessel (2 mL) connected to a quartz cell (1 cm
optical path length) fixed to a gas/vacuum manifold by a glass joint.
After the phosphate buffer (pH 7.4) solution of trans-[Ru(NH3)4(NO2)P-
(OEt)3]+ was degassed, the hemeprotein (hem) was introduced into the
round-bottom vessel, and the system was deaerated with four vacuum-
argon cycles. The absorption spectrum of the aqueous solutions of the
complex was measured and photolyzed with 355 nm light for 6 min.
When the irradiation was stopped, the hem was mixed, and the
subsequent thermal reaction was followed by changes in the UV-vis
absorption spectrum of the protein. Products were identified by
comparing their UV-vis spectra with the spectra of the (NO)FeII/III
hem adduct.22,23 The absorption spectrum of hem in the presence of
the complex was stable in the dark for long periods.
atoms
dist
atoms
angle
Ru-N5
Ru-N4
Ru-N2
Ru-N3
Ru-N1
Ru-P1
P1-O2
P1-O1
P1-O3
O1-C11
O2-C21
O51-N5
O3-C31
2.096(3)
2.141(2)
2.133(2)
2.141(2)
2.142(2)
2.217(1)
1.615(2)
1.614(2)
1.604(2)
1.452(4)
1.451(3)
1.251(3)
1.462(4)
N5-Ru-P1
N4-Ru-P1
N2-Ru-P1
N3-Ru-P1
N1-Ru-P1
O51-N5-O52
O51-N5-Ru
O52-N5-Ru
178.47(7)
93.08(7)
94.01(7)
95.01(7)
92.39(7)
117.7(2)
120.9(2)
121.4(2)
exchange-correlation functional24 known as B3LYP at the 6-31(d) level
of theory for H, C, N, O, and P, and 3-21 g(d) with LanL2DZ pseudo
potential25 for Ru. The analytical evaluation of the energy second
derivative matrix in Cartesian coordinates (Hessian matrix) at the same
level of approximation confirmed the nature of minima of the potential
surface points associated with the optimized structures.
Time-dependent density functional theory26-28 (TD-DFT) allowed
the computation of excitation energies, oscillator strengths, and
composition determinants. TD-DFT calculations were performed using
the B3LYP hybrid functional and the 6-31++G(d,p) basis set for H,
C, N, O, and P, and an effective core potential LanL2DZ for Ru. The
excited state compositions were described as a linear combination of
singly excited Slater determinants; their relative weights are represented
by the normalized squared coefficients in the linear combination.
Results and Discussion
Molecular Structure. Table 1 shows the selected interatomic
distances. The mean Ru-NH3 distance is determined as 2.14(4)
Å, which is in agreement with that (2.13 ( 0.01 Å) in trans-
[Ru(NH3)5NO2]Cl29 and that (2.10 ( 0.06 Å) in trans-[Ru(NH3)4-
(NO)(nic)](SiF6)3‚2H2O.12 The Ru-P distance [2.2171(8) Å]
is shorter than that (2.366 Å) in trans-[Ru(NH3)4(P(OEt)3)2]-
(PF6)2.16 The P-Ru-N(NO2) bond angle is 178.47(7)°, in-
dicating that the P-Ru-N(NO2) bond is almost linear. The
O-N-O angle of the NO2 in the moiety trans-[Ru(NH3)4(NO2)-
P(OEt)3](PF6) is 117.7(2)°, similar to those obtained for
[Ru(NO2)(PMe3)3(trpy)](ClO4) (114.8°), trans-[Ru(NH3)5NO2]-
Cl (113.2°), and [Ru(trpy)(NC5H4NdNC6H4(8-Me))NO2]ClO4‚
C6H6 (117.6°).29,30
Molecular Orbital Results. For DFT calculations, the
(P(OEt)3)P-Ru-N(NO2) vector is defined as x, while z and y
lie close to the Ru-NH3 vectors (Figure 1, inset).
(24) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M.
A.; Cheeseman, J. R.; Zakrzewski, V. G.; Montgomery, J. A., Jr.; Stratmann,
R. E.; Burant, J. C.; Dapprich, S.; Millam, J. M.; Daniels, A. D.; Kudin,
K. N.; Strain, M. C.; Farkas, O.; Tomasi, J.; Barone, V.; Cossi, M.; Cammi,
R.; Mennucci, B.; Pomelli, C.; Adamo, C.; Clifford, S.; Ochterski, J.;
Petersson, G. A.; Ayala, P. Y.; Cui, Q.; Morokuma, K.; Malick, D. K.;
Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Cioslowski, J.; Ortiz,
J. V.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.;
Gomperts, R.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng,
C. Y.; Nanayakkara, A.; Gonzalez, C.; Challacombe, M.; Gill, P. M. W.;
Johnson, B. G.; Chen, W.; Wong, M. W.; Andres, J. L.; Head-Gordon,
M.; Replogle, E. S.; Pople, J. A. Gaussian 98, revision A.6; Gaussian,
Inc.: Pittsburgh, PA, 1998.
Computational Details. All calculations were performed using the
Gaussian 98 suite of programs.24 The starting molecular geometries
were obtained at the UHF/3-21G level of theory. The final molecular
geometry optimizations were performed using the Kohn-Sham density
functional theory (DFT) with the Becke three-parameters hybrid
(25) Wadt, W. R.; Hay, P. J. J. Chem. Phys. 1985, 82, 284-298.
(20) Sheldrick, G. M. SHELX93, Program for Crystal Structure Determination;
University of Gottingen: Gottingen, Germany, 1993.
(21) Johson, C. K. ORTEPII Report ORNL-5138; Oak Ridge National Labora-
tory: Oak Ridge, TN, 1976.
(22) Hoshino, M.; Ozawa, K.; Seki, H.; Ford, P. C. J. Am. Chem. Soc. 1993,
115, 9568-9575.
(23) Hoshino, M.; Maeda, M.; Konishi, R.; Seki, H.; Ford, P. C. J. Am. Chem.
Soc. 1996, 118, 5702-5707.
(26) Becke, A. D. J. Chem. Phys. 1993, 98, 5648-5652.
(27) Casida, M., Chong, D. P., Eds. Recent AdVances in Density Functional
Methods; World Scientific: Singapore, 1995; Vol. 1, p 155.
(28) Casida, M. E.; Jamorski, K. C.; Casida, D. R.; Salahub, D. R. J. Chem.
Phys. 1998, 108, 4439-4449.
(29) Bottomley, F. J. Chem. Soc., Dalton Trans. 1972, 2148-2152.
(30) Leising, R. A.; Kubow, S. A.; Churchill, M. R.; Buttrey, L. A.; Ziller, J.
W.; Takeuchi, K. J. Inorg. Chem. 1990, 29, 1306-1312.
9
2548 J. AM. CHEM. SOC. VOL. 126, NO. 8, 2004

Radicals of trans-[Ru(NH ) (NO2)P(OEt)3](PF6)
A R T I C L E S
3 4
to the lowest π* orbital of the nitro ligand. This conclusion is
in agreement with the results from the MO calculations. The
lowest energy band, 3.66 eV (339 nm), is very likely a charge-
transfer transition from the HOMO localized in the metal to
the LUMO orbital localized in the nitro ligand. The second
MLCT transition was calculated at 3.74 eV (331 nm) represent-
ing mainly a HOMO-3 to LUMO transition. The second
transition is weaker, because the magnitude of its transition
electric dipole moment is smaller (5.37 × 10-2 au) as compared
to the former one (6.15 × 10-2 au).
The excited singlet state corresponding to a HOMO-LUMO
excitation is calculated to be at 3.66 eV above the ground state,
which is in remarkable agreement with the experimental energy
of 3.71 eV.
Apart from the CT transitions, the nitro complex also
possesses LF transitions at lower energies, as their low intensities
are obscured by the intense CT transitions. In the MO diagrams
of trans-[Ru(NH3)4(NO2)P(OEt)3]+, the transitions from HOMO
to LUMO+2 (11% py Ru, 9% pz Ru, 21% dz2 Ru, 8% dx2-y2
Ru) with an energy of 3.05 eV (406 nm) can be considered to
be LF transitions.
Photochemistry. The photolysis of trans-[Ru(NH3)4(NO2)P-
(OEt)3]+ (6 × 10-4 mol L-1) in CH3CN with 355 nm light led
to a progressive depletion of the characteristic absorption band
at 308 nm (Figure 4) concomitantly with a blue shift to 292
nm. This new band profile is similar to that exhibited17 by trans-
[Ru(NH3)4(H2O)P(OEt)3]2+ (λmax) 292 nm, ꢀ ) ∼8 × 102
mol-1 L cm-1) in CH3CN. Isosbestic points were observed,
indicating that there is no spectrally significant buildup of
intermediates and any potential secondary photolysis does not
lead to measurable spectral changes. This observation was
confirmed by the direct photolysis of the trans-aquophosphite
species synthesized thermally.16 No spectral changes were found
after a long photolysis period of trans-[Ru(NH3)4(H2O)P-
(OEt)3]2+ in CH3CN.
The presence of trans-[Ru(NH3)4(H2O)P(OEt)3]2+ in the
photolyzed solution was confirmed through 31P NMR measure-
ments. The irradiation of trans-[Ru(NH3)4(NO2)P(OEt)3]+ in
CH3CN for 30 min at room temperature afforded a well-resolved
31P NMR spectrum with one signal at 147 ppm, which was
assigned to trans-[Ru(NH3)4(H2O)P(OEt)3]2+ (Figure 5).
Nitric oxide was detected in the analysis of partially photo-
lyzed solutions (10-40% conversion) through differential pulse
polarography (DPP), Figure 6. The anodic pulse observed at
0.80 V vs SCE was assigned to the oxidation of the radical
produced. This peak was absent in solutions of trans-[Ru(NH3)4-
(NO2)P(OEt)3]+ kept in the dark. The quantification of nitric
oxide was performed using the NO sensor electrode. In such
photolysis experiments, the NO concentration was determined
electrochemically after ∼20 min of photolysis of the trans-[Ru-
(NH3)4(NO2)P(OEt)3]+ (∼6.39 × 10-4 mol L-1) in CH3CN. The
data collected indicated that 100 nmol L-1 of NO was released
per mol of photo converted complex.
Figure 2. Relevant parts of the molecular orbital diagram of trans-[Ru-
(NH3)4(NO2)P(OEt)3]+.
The quantum chemical calculations gave distances for
Ru-N(NH3) of 2.20 Å, Ru-N(NO2) of 2.06 Å, and Ru-P-
(P(OEt)3) of 2.31 Å, in very good agreement with those obtained
from single-crystal X-ray data. The calculated N-O distances
from NO2- are 1.24 Å. The complex has C1 symmetry with P,
Ru, and N (all on the x axis) of the axial nitro ligand in the
-
same plane of symmetry. The NO2 group lies on a plane
parallel to the x axis and bisects the y and z axes, that is, the
NH3 axes.
Molecular Orbital Analysis. The frontier orbitals were
obtained using the B3LYP functional. The HOMO has pre-
dominantly (60%) nonbonding dyz character, while HOMO-1
has a nonbonding character with main contributions of (42%)
dxy and (23%) dxz. The LUMO has 72% of NO2 π* character
(pz orbitals of N and both O). Interestingly, because the molecule
belongs to the C1 group, the LUMO+1 and LUMO+2 MOs
have s, px, py, pz, dz2, and dx2-y2 contributions. The relative
energies for HOMO and LUMO are -8.92 and -4.13 eV,
respectively. It is important to notice that density functional
methods usually give small HOMO-LUMO gaps. The next two
empty MOs LUMO+1 and LUMO+2 are 0.74 and 0.85 eV
higher in energy as compared to the LUMO energy. The
HOMO-1 and HOMO-2 MOs are 0.28 and 0.38 eV below
the HOMO energy. Because the energy difference between
LUMO+1 and LUMO+2 is near 0.1 eV, the two states may
be considered as nearly degenerated, which is also valid for
HOMO-1 and HOMO-2 MOs. The MO diagrams of trans-
[Ru(NH3)4(NO2)P(OEt)3]+ are presented in Figure 2.
Electronic Spectrum. Figure 3 clearly shows that the nature
of the solvent has a great influence on the position and the
relative intensities of the absorption band of trans-[Ru(NH3)4-
(NO2)P(OEt)3]+. In CH3CN, the absorption spectrum shows a
weak shoulder at about 290 nm and a band maximum at 308
nm. Changing the solvent from CH3CN to phosphate buffer,
pH ) 9.7, results in a shift of the absoption maxima to longer
wavelengths. At the same time, the intensity of the higher-lying
absorption band increases with respect to that of the band of
the lower one. On the basis of these effects, the bands are
assigned as MLCT charge-transfer transitions from the metal
to the nitro ligand. Indeed, these two close absorptions might
belong to MLCT transitions from different metal-dπ orbitals
It was possible to infer that the presence of nitric oxide was
the result of the secondary photolysis of free nitrite. Additional
support was provided by a photochemical study31 which showed
that the photolysis of the nitrite in aqueous solution in the 200-
400 nm region leads to the production of both a nitrosyl radical
(31) Mack, J.; Bolton, J. R. J. Photochem. Photobiol., A: Chem. 1999, 128,
1-13.
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Carlos et al.
Figure 3. Absorption spectra of trans-[Ru(NH3)4(NO2)P(OEt)3]+ in both CH3CN (A) and phosphate buffer pH 9.3 (B).
Figure 4. Spectral changes accompanying the photolysis of trans-[Ru(NH3)4(NO2)P(OEt)3]+ in CH3CN, λirr ) 355 nm.
and an oxygen radical anion. The recombination is likely to
occur due to the high reactivity of the species.31-33 The
observance of nitric oxide suggests that where aprotic solvents
are present the oxygen radical anion is trapped, probably by
the solvent, allowing for the observation of NO. Therefore, the
observation of trans-[Ru(NH3)4(H2O)P(OEt)3]2+ as the primary
photolysis product in CH3CN would be consistent with the
reaction Scheme 1.
The spectral changes were totally different when trans-[Ru-
(NH3)4(NO2)P(OEt)3]+ was photolyzed in phosphate buffer pH
9.0. The irradiation at 355 nm of a solution containing ∼7 ×
10-4 mol L-1 complex led to spectral variations, which were
in accordance with those found in CH3CN for a time period
corresponding to about 25% conversing, that is, a continuous
decrease in the absorbance around 330 nm and a shift in the
maximum absorption to 316 nm (Figure 7). However, when
the irradiation was stopped and the sample was allowed to
remain in the dark for 20 min, a final spectrum was observed,
which was similar to that of trans-[Ru(NH3)4(H2O)P(OEt)3]3+
in the same solvent (λmax ) 285 nm, ꢀ ≈ 800 mol-1 L cm-1).
The analysis was based on the changes in a photolyzed solution
of trans-[Ru(NH3)4(NO2)P(OEt)3]+ using 31P NMR spectros-
copy. The spectral changes (Figure 8) indicate that the disap-
pearance of the trans-[Ru(NH3)4(NO2)P(OEt)3]+ signal is
accompanied by the appearance of a new signal at 147 ppm
characteristic of 31P in trans-[Ru(NH3)4(H2O)P(OEt)3]2+, as well
as of an unidentified species with a 31P signal at 143 ppm. A
signal for a free phosphite ligand was also observed at 9 ppm.
The presence of a paramagnetic ruthenium species was
detected in the EPR spectra of photolyzed trans-[Ru(NH3)4-
(32) Meyrstein, D. Acc. Chem. Res. 1978, 11, 43-48.
(33) Stanbury, D. M. AdV. Inorg. Chem. 1989, 33, 69-80.
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Radicals of trans-[Ru(NH ) (NO2)P(OEt)3](PF6)
A R T I C L E S
3 4
Figure 5. 31P NMR spectrum of trans-[Ru(NH3)4(NO2)P(OEt)3]+ in CH3CN after 30 min of photolysis.
Figure 6. Differential pulse voltammogram of trans-[Ru(NH3)4(NO2)P(OEt)3]+ complex in CH3CN at the glassy-carbon electrode with platinum wire as an
auxiliary electrode and SCE as the reference electrode. (A) Before photolysis, (B) after photolysis.
Scheme 1. Primary Step during the 355 nm Photolysis of
alkaline solutions, Ru(II) and Ru(VI) are expected to be more
stable than Ru(III). The EPR spectrum of trans-[Ru(NH3)4-
trans-[Ru(NH3)4(NO2)P(OEt)3]+
(H2O)P(OEt)3]3+ at pH 9.0 showed a more complicated pat-
tern17 than at pH 2.0, as a result of the mixture of at least
three reaction products. One of the components was identi-
fied as a paramagnetic species,18 which was also found in the
mixture of photoreaction products after irradiation. Unfortu-
nately, the identity of this species has not been unequivocally
established. By analogy with the literature data,17,18 trans-[Ru-
(NH3)4(H2O)P(OEt)3]3+ may be assigned as a precursor of this
paramagnetic species.
(NO2)P(OEt)3]+ solutions. The (frozen solution) EPR spec-
trum observed ca. 20 min after irradiation shows a small
amount of an unidentified species at g ) 2.06, which is
neither stable nor frequently evident during the experiments
(Figure 9). This observation would suggest that, in the decay
processes, the autoxidation or diffusion from the solution may
be occurring.
As previously reported, the stability of trans-[Ru(NH3)4-
(H2O)P(OEt)3]3+ is highly pH-dependent.17,18 At pH > 11, a
disproportionate reaction is suggested to occur, yielding Ru(II)
and Ru(VI) as the major entities.18 It is also argued that, in
The results of our experiments strongly suggest that trans-
[Ru(NH3)4(H2O)P(OEt)3]2+ is the primary reaction product in
the photochemistry of trans-[Ru(NH3)4(NO2)P(OEt)3]2+. It is
also stable unless a very reactive radical is present to trap this
cation and initiate, probably through a chain reaction, its
oxidation with a consequent decomposition through alterations
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Carlos et al.
Figure 7. Spectral changes of the absorption spectrum accompanying the photolysis of trans-[Ru(NH3)4(NO2)P(OEt)3]+ in phosphate buffer pH 9.3, λirr
355 nm.
)
Figure 8. 31P NMR spectrum of trans-[Ru(NH3)4(NO2)P(OEt)3]+ in borax-buffered D2O (pH 9.0) after 20 min of photolysis.
of the oxidized complex. The most obvious oxidant is a
hydroxide radical, as it is produced during the photolysis of
nitrite ion in alkaline solutions.31
measurements. The EPR signals of the ascorbyl radical disap-
peared in the dark. On the contrary, the signals increased when
the nitrite ion was replaced by the trans-[Ru(NH3)4(NO2)P-
(OEt)3]+ complex.
To provide support for this hypothesis, ascorbic acid was
introduced as a trap for any key intermediate produced during
the photolysis of trans-[Ru(NH3)4(NO2)P(OEt)3]2+. As is de-
scribed in the literature,34 the ascorbate anion, AH-, would be
The irradiation of trans-[Ru(NH3)4(NO2)P(OEt)3]+ (0.028 mol
L-1) in the presence of ascorbic acid (0.057 mol L-1) inside
the EPR cavity resulted in a continuous decrease of the ascorbyl
radical signal, as a function of time, Figure 10.
•
oxidized by either OH• or NO2 radical produced by the
The ascorbyl radical acts as a trap for OH•, NO2 , and NO•
•
photolysis of nitrite in aqueous solution (Scheme 2).
In control experiments, nitrite (0.25 mol L-1) was irradiated
with 355 nm light in the presence of ascorbic acid (0.01 mol
L-1). There was an increase in the formation of the ascorbyl
radical during irradiation, as is confirmed by in-situ EPR
and as a reductor for Ru(III), thereby preventing the gene-
ration of new species capable of initiating a chain reaction
(34) Bilski, P.; Chignell, C. F.; Szychlinski, J.; Borkowsky, A.; Olesky, E.;
Reszka, K. J. Am. Chem. Soc. 1992, 114, 549-556.
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Radicals of trans-[Ru(NH ) (NO2)P(OEt)3](PF6)
A R T I C L E S
3 4
Figure 9. EPR spectrum of trans-[Ru(NH3)4(NO2)P(OEt)3]+ complex in phosphate buffer pH 9.0 frozen after 20 min of irradiation at 355 nm.
Figure 10. EPR spectrum of trans-[Ru(NH3)4(NO2)P(OEt)3]+ complex in phosphate buffer pH 9.0 in the presence of ascorbic acid frozen after 20 min of
irradiation at 355 nm. (A) Laser off, (B) laser on for 1 min, (C) laser on for 5 min, (D) laser on for 15 min.
Scheme 2. Reaction of the Radicals Produced during NO2-
Photolysis in the Presence of Ascorbic Acid
From these results, it is evident that the rate for hydroxide
abstraction by Ru(II) would be much faster than the rate for
radical recombination34 (OH• + NO2• f HNO2; k ) 1.0 × 1010
L mol-1 s-1).
The fact that trans-[Ru(NH3)4(H2O)P(OEt)3]2+ is produced
during the photolysis of trans-[Ru(NH3)4(NO2)P(OEt)3]+ either
in CH3CN or in aqueous solution indicates that the principal
deactivation pathway of the initial MLCT state is an internal
3
conversion to a common reactive state, presumably the LF*
excited state to which ligand photolabilization is attributed. It
is likely that the secondary photolysis of free nitrite occurs. In
aqueous solution, this deactivation pathway leads to the forma-
tion of a hydroxide radical, which in the presence of trans-
[Ru(NH3)4(H2O)P(OEt)3]2+ would initiate a chain reaction. The
observation of trans-[Ru(NH3)4(H2O)P(OEt)3]3+ as a reaction
product in aqueous solution suggests the following reaction
sequence (Scheme 3).
leading to the depletion of trans-[Ru(NH3)4(H2O)P(OEt)3]2+
.
In addition, the recombination reaction of Ru(III) and AH-
and/or A•- as a termination step of a chain reaction would also
be consistent with a decrease in the ascorbyl radical signals
during the photolysis of trans-[Ru(NH3)4(NO2)P(OEt)3]2+
.
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Figure 11. Spectral changes accompanying the formation of nitrosylmyoglobin (NO)Mb(II) from the thermal reaction of Mb(III) and trans-[Ru(NH3)4-
(NO2)P(OEt)3]+ complex, after 15 min of photolysis, in phosphate buffer pH 7.4. Inset: Spectral changes observed for Cyt(III) during the thermal reaction
with photolyzed trans-[Ru(NH3)4(NO2)P(OEt)3]+ complex in phosphate buffer pH 7.4.
Scheme 3. Mechanism Proposed for the Reaction of
reductive nitrosylation of Mb(III), as the spectrum of (NO)-
trans-[Ru(NH3)4(NO2)P(OEt)3]+ upon 355 nm Light Irradiation
Mb(II) produced by the reaction of Mb(II) and NO also has
absorption peaks22 at 420 (ꢀ ) 1.29 × 105 mol L-1 cm-1), 545,
and 578 nm.
Hemoglobin. The absorption spectral changes of hemoglo-
bin(III), λabs ) 403 nm, in the presence of a photolyzed solu-
tion of trans-[Ru(NH3)4(NO2)P(OEt)3]+ were studied in phos-
phate solutions. The NO adduct of hemoglobin, Hb(III), λabs
)
417 nm in the Soret band region and 532 and 563 nm in the
Q-band region,22 initially produced was gradually changed to
(NO)Hb(II), 417, 542, and 570 nm. The mechanism involved
in the formation of (NO)Hb(II) appears to be analogous to that
of (NO)Mb(II) formation from Mb(III).
The presence of nitric oxide in this photoreaction is demon-
strated by the experiments of heme proteins nitrosylation
described below.
Reductive Nitrosylation. The direct reactions of NO with
iron metalloproteins are the most important biological reactions
in which NO participates.3 For instance, the scavenging of NO
by oxymyoglobin makes the heme protein a protector of cellular
respiration through this function, where the otherwise slow
oxidation of NO to nitrate is catalyzed efficiently.35 NO also
binds to iron in other heme proteins such as myoglobin,
cytochromes, and hemoglobin.3 In this context, attention has
been devoted to the absorption spectroscopic studies on the
interaction of heme proteins with NO generated from the
photolyzed solution of trans-[Ru(NH3)4(NO2)P(OEt)3]+. These
studies are described below.
Cytochrome C. An anaerobic solution of trans-[Ru(NH3)4-
(NO2)P(OEt)3]+, 0.006 g in 5 mL, was photolyzed with 355
nm light for 6 min. When the irradiation was stopped, cyto-
chrome C, cyt(III), 0.002 g, was introduced into the cu-
vette. Notably, the spectrum measured over a period of min-
utes after the initiation of the reaction was identical to that of
authentic reduced cytochrome C, cyt(II) (413, 518, and 549
nm).22,23 The subsequent spectral changes which exhibit isos-
bestic points are interpreted in terms of (NO)cyt(II) formation.
Although preliminary, it is interesting to stress that, in our
experiment, cytochrome C, which is structurally quite different
from Mb(III) and Hb(III), reacts faster with photolyzed trans-
[Ru(NH3)4(NO2)P(OEt)3]+ than do Mb(III) and Hb(III).
Myoglobin. The electronic spectrum of myoglobin, Mb(III),
in phosphate buffer pH 7.4 exhibits absorption maxima at 408
nm (ꢀ ) 1.5 × 105 mol L-1 cm-1) and 500 nm (ꢀ ) 1.0 × 104
mol L-1 cm-1).22 When the Mb(III) was mixed to the photolyzed
solution of trans-[Ru(NH3)4(NO2)P(OEt)3]+, Figure 11, new
bands appeared at 418 nm in the Soret band region and at 536
and 567 nm in the Q-band region, because of the formation22,23
of (NO)Mb(III). After a few minutes, the latter bands were red-
shifted to 545 and 578 nm, while the one at 418 nm decreased
to 420 nm. The overall spectral change was interpreted as the
Conclusions
The absorption spectrum of trans-[Ru(NH3)4(NO2)P(OEt)3]+
is dominated by an intense MLCT transition band, but irradiation
at 355 nm leads to photosubstitution processes typical of LF
photochemistry. The irradiation wavelength of 355 nm is very
close in energy to that of the lower energy ligand field band
determined by TD-DFT calculations (406 nm), which are
ascribed to a dz2 r dxz,dyz transition. In the C4V point group, the
lowest LF states involve the population of σ* states directed
along the O2N-Ru-P(OEt)3 axis. The nitro labilization in the
(35) Moller, J. K. S.; Skibsted, L. H. Chem. ReV. 2002, 102, 1167-1178.
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Radicals of trans-[Ru(NH ) (NO2)P(OEt)3](PF6)
A R T I C L E S
3 4
Ru(II) complex is consistent with the electron-withdrawing
nature of this ligand, which leads to enhanced positive charge
density in the nitro ligand, thus lowering its σ-donor capability
relative to the phosphite ligand and leading to increased lability.
The photochemical behavior of trans-[Ru(NH3)4(NO2)P-
(OEt)3]+ in the presence of hemeproteins in physiologic pH
displays reactivity properties relevant to the mechanisms of NO
reactions with Fe(III) proteins in vivo and proves that trans-
[Ru(NH3)4(NO2)P(OEt)3]+ is a potential photochemical delivery
agent of NO to biological targets. Furthermore, the trans-[Ru-
(NH3)4(H2O)P(OEt)3]2+ species produced would act as an
antioxidant, as it is efficiently trapped by reactive hydroxyl
radicals. It is important to note that as far as we know in vitro
generation of a (NO)hem adduct from the photochemical
activation of a metal transition complex has not been previously
reported.
Acknowledgment. The authors would like to acknowledge
FAPESP, CNPq, and CAPES for the grants and fellowships
given to the research. They are also indebted to Prof. Bruce R.
McGarvey for his helpful discussions and Prof. Peter C. Ford
for reading and commenting on this manuscript.
Supporting Information Available: X-ray crystallographic
data for structure determinations of trans-[Ru(NH3)4(NO2)P-
(OEt)3]+ (CIF). This material is available free of charge via
the Internet at http://pubs.acs.org.
JA0373263
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