Photosensitized production of nitric oxide and peroxynitrite from a carbon-bound diazenium diolate and 2-methyl-2-nitrosopropane

Article abstract of DOI:10.1016/j.jphotochem.2016.07.023

The photosensitized generation of nitric oxide from alanosine (3-(hydroxynitrosoamino)-D,L-alanine) by aluminum phthalocyanine tetrasulfonate (AlPcS4) is reported. While nitric oxide (NO) is obtained in nitrogen-saturated solutions, evidence suggest that both NO and peroxynitrite are produced in air-saturated solutions. Enhancement of NO production occurs in the presence of ubiquinone-0. These observations support the idea that NO is produced by the photosensitized oxidation of alanosine. Both NO and peroxynitrite are detected during photoirradiation of AlPcS4 in the presence of 2-methyl-2-nitrosopropane (MNP) and hypoxanthine (HX), but not in the absence of HX, in air-saturated solutions, thus implying that HX is acting as sacrificial electron donor, thus promoting superoxide formation.

Full text of DOI:10.1016/j.jphotochem.2016.07.023

Journal of Photochemistry and Photobiology A: Chemistry 330 (2016) 79–85
Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology A:
Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
Photosensitized production of nitric oxide and peroxynitrite from a
carbon-bound diazenium diolate and 2-methyl-2-nitrosopropane
*
Pedro Sanchez-Cruz, Antonio E. Alegria
Department of Chemistry, University of Puerto Rico, PO Box 860, Humacao, PR 00792, United States
A R T I C L E I N F O
A B S T R A C T
Article history:
The photosensitized generation of nitric oxide from alanosine (3-(hydroxynitrosoamino)-D,L-alanine) by
Received 14 April 2016
Received in revised form 20 July 2016
Accepted 25 July 2016
Available online 27 July 2016
aluminum phthalocyanine tetrasulfonate (AlPcS4) is reported. While nitric oxide (NO) is obtained in
nitrogen-saturated solutions, evidence suggest that both NO and peroxynitrite are produced in air-
saturated solutions. Enhancement of NO production occurs in the presence of ubiquinone-0. These
observations support the idea that NO is produced by the photosensitized oxidation of alanosine. Both NO
and peroxynitrite are detected during photoirradiation of AlPcS4 in the presence of 2-methyl-2-
nitrosopropane (MNP) and hypoxanthine (HX), but not in the absence of HX, in air-saturated solutions,
thus implying that HX is acting as sacrificial electron donor, thus promoting superoxide formation.
ã 2016 Elsevier B.V. All rights reserved.
Keywords:
Carbon-bound diazenium diolate
Alanosine
Photosensitization
Oxidation
Nitric oxide
Peroxynitrite
Photodynamic therapy
1. Introduction
with transition metals and heme-containing proteins [8]. Due to
the reactive nature of gaseous NO, its short half-life, instability
Nitric oxide (NO) is an endogenously produced molecule that
has multiple roles in physiological processes, including angiogen-
esis, wound healing, neurotransmission, smooth muscle relaxa-
tion, and inflammation [1]. Nitric oxide’s action on physiology is
highly dependent on location, source, and concentration [2]. It is
produced in vivo by NO synthase (NOS). Low nanomolar NO
concentrations are produced by eNOS and nNOS to promote
vasodilation and neurotransmission, respectively [3]. The iNOS
form is capable of producing micromolar levels of NO, often
responding to infection and inflammation [1]. In the presence of
superoxide (O2ꢀ), NO will react to form peroxynitrite (ONOOꢁ), an
even greater oxidant involved in the inflammatory response [4].
Peroxynitrite causes apoptotic or necrotic cell death through
nitration of tyrosine residues in proteins, lipid peroxidation,
oxidation of critical thiols, DNA strand breaks, NAD depletion and
thus energy failure [5]. NO is also a wound healing promoting
agent and, due to its antibacterial activity, it is a promising agent
for reducing implant-associated infections and promoting tissue
regeneration in orthopedic procedures [6,7].
during storage, and potential toxicity, including its influence on the
systemic blood pressure, chemical strategies for NO storage and
release have been developed in an effort to use NO’s pharmaco-
logical potential. Several ways of NO release to tissues have been
developed. Diazeniumdiolates (1-amino-substituted diazen-1-
ium-1,2-diolate, i.e. NONOates) and S-nitrosothiols represent the
two most diverse NO donor classes [9,10]. Other classes are organic
nitrates and metal nitrosyl compounds such as sodium nitroprus-
side and potassium nitrosylpentachlororuthenate [9].
The release of NO from several nanocarriers have been
developed to avoid systemic NO side effects while transporting
the NO source to the selected tissue [11]. The selective delivery of
NO to tissues in adequate concentrations is a developing area of
research. These include polymeric nanoparticles, micelles, den-
drimers, nanogels, gels, gold nanoparticles, silica nanoparticles and
liposomes [11]. The possibility of releasing NO before reaching the
tissue site is still a major problem in particle-based systems. In
addition, the rate of release of NO at the tissue site using those
systems is difficult to be controlled and those where controlled
release of NO is observed are mostly metal-based nanoparticle
systems containing transition metals with the potential toxicity of
those remaining to be tested.
However, nitric oxide has a short half-life (<1 s) in the presence
of oxygen and hemoglobin in vivo, arising from its high reactivity
One way of selectively release NO at the needed tissue is to use
tissue-penetrating light (wavelengths in the near infrared region,
NIR) to activate NO release from molecules. A recent technique,
* Corresponding author.
E-mail address: antonio.alegria1@upr.edu (A.E. Alegria).
http://dx.doi.org/10.1016/j.jphotochem.2016.07.023
1010-6030/ã 2016 Elsevier B.V. All rights reserved.

80
P. Sanchez-Cruz, A.E. Alegria / Journal of Photochemistry and Photobiology A: Chemistry 330 (2016) 79–85
using a 2 photon laser irradiation where NIR photons are added to
produce more energetic photons, and NIR-to-visible up-conver-
sion, which are able to release NO from NO-containing molecules
and has been developed and used in NO-containing nanoparticles
[12–14]. This technique permits the use of longer, tissue-
penetrating wavelengths for the photochemical release of NO at
the selected tissue site. The use of liposomes for photodelivering
NO from NO-containing chromium complexes has also been
reported, where NO is detected outside the liposome [15].
However, a non-tissue-penetrating light wavelength was used
[15]. The technical problem to overcome is that those photo-
controllable NO donors, where all of them contain transition
metals, may exhibit systemic toxicity due to release of transition
metal ions. In addition, those systems do not generate peroxyni-
trite, a species which should enhance the toxic activity of NO.
Cupferron, a carbon-bound diazenium diolate, is able to
produce nitric oxide photochemically [16] and upon enzymatic
oxidation [17]. A natural product with carbon-bound diazenium
diolate structure, without the potential carcinogenicity of cupfer-
ron [18], is alanosine, Fig. 1. Furthermore, the possibility of
generating carcinogenic nitrosamines, as could occur after
photolysis of nitrogen-bound diazeniumdiolate ions [19], has
not been reported for carbon-bound diazeniumdiolates. Since
other NO-containing compounds release NO by photosensitization
[20], we were prompted to test the photosensitized generation of
NO from this type of compound. In this work we report the
photosensitized release of NO from alanosine using NIR radiation.
Evidence supports the generation of peroxynitrite from air-
saturated dye-alanosine solutions and from air-saturated 2-
methyl-2-nitrosopropane (MNP) solutions containing a sacrificial
electron donor. Although the photosensitized generation of NO
from MNP has been reported previously [20], evidence suggesting
the photosensitized production of peroxynitrite by MNP in the
presence of the sacrificial electron donor, hypoxanthine (HX), is
described here. In the present work we have used MNP to contrast
the behavior of alanosine. The photosensitized production of NO
could be used in photodynamic therapies of malignancies, where
NO or peroxynitrite are used as toxic agents, and where
nanoparticle carriers containing both the NO source and the
photosensitizer, are transported to the desired tissue.
were purchased from Sigma-Aldrich Co. All solutions were
prepared in phosphate buffer and used the same day. Deionized
and Chelex-treated water was used in the preparation of all stock
and sample solutions. Chelex treatment of water and buffer was
monitored using the ascorbate test, as described by Buettner [21].
Care was always taken to minimize exposure of solutions to light.
2.2. Methods
2.2.1. Sample irradiation for EPR analysis
The NO probe, carboxy-PTIO, was used to detect NO formation
from the production of the carboxy-PTI EPR spectrum, as reported
previously [22–24]. Air- or N2-saturated samples containing
AlPcS4 (with absorbance of 1 at 675 nm), alanosine (or MNP, in
the presence and absence of HX), in the presence or absence of
ubiquinone-0 and carboxy-PTIO in 50 mM phosphate buffer (pH
7.4) were irradiated at 675 nm in a 1 cm light path Pyrex cuvettes
with continuous stirring for different periods of time. At the end of
each period, samples were then transferred into N2- or air-
saturated EPR flat quartz cells (60 ꢂ10 ꢂ 0.25 mm) and placed in
the EPR instrument cavity for analysis. A 1000 W xenon arc lamp
coupled to a Spectral Energy GM 252 high-intensity grating
monochromator with a bandwidth of ꢃ 20 nm was used as the
irradiation source. EPR spectra were recorded on a Bruker ER-200D
spectrometer at 100 kHz magnetic field modulation. EPR line
intensities were determined from the peak-to-peak derivative
amplitudes times the square of the peak-to-peak widths.
2.2.2. Sample irradiation in the NO electrode chamber
Nitric oxide production rates were monitored using a NO-
specific electrochemical probe (ISO-NOP) inserted in a thermo-
stated NO chamber (World Precision Instruments, Sarasota, FL) at
37 ꢄC. The chamber was either saturated with air or purged with
high purity nitrogen followed by injection of 1.00 mL of an air- or
nitrogen-saturated solution containing from 0 to 1 mM alanosine
or 0–3 mM MNP, 10 mM AlPcS4 and 0 or 500 mM UBQ-0 in 50 mM
phosphate buffer (pH 7.4). This was followed by immediate
exclusion of all gas bubbles out of the sample, through the
chamber capillary. The sample was continuously stirred using a
spinning bar. Data acquisition was started before irradiation. The
sample was then irradiated at 670 nm using a B&W Tek diode
laser with a constant power of 255 mW. Basal voltage was
calibrated to zero every day. Voltage output corresponding to a
2. Materials and methods
2.1. Materials
20 mM NO solution was checked every day, and the electrode
membrane was replaced in case there was no agreement with
previous outputs within 10%. The electrode was calibrated daily
with known concentrations of NaNO2 by reacting this salt with KI
in sulfuric acid medium. NO production data were collected in a
computer, and the initial rates of NO consumption (RNO) were
measured. RNO values reported are averages of 3 determinations
for each type of sample.
Alanosine 3-(hydroxynitrosoamino)-D,L-alanine, Fig. 1, was
obtained from the NCI DTP Repository (Rockville, MD). The dye
aluminum phthalocyanine tetrasulfonate (AlPcS4) was purchased
from Frontier Scientific. The compounds ubiquinone-0 (UBQ-0),
ferricytochrome c, MNP, HX, L-tyrosine, carboxy-PTIO, superoxide
dismutase (SOD, from bovine erythrocytes) and 3-nitrotyrosine
2.2.3. Peroxynitrite formation
Peroxynitrite formation was detected indirectly by its reaction
with
L
-tyrosine to produce 3-nitrotyrosine, as described previously
-tyrosine were
[25]. For this purpose, micromolar amounts of
L
included in the air-saturated samples to be irradiated and its nitro-
substituted product detected at 274 nm using HPLC. HPLC analyses
were performed using a HP Zorbax SB-C18 (4.6 ꢂ 250 mm) column
and eluted with a solvent mixture of 95% ammonium acetate (pH
4.7) and 5% methanol. An Agilent 1100 analytical HPLC system with
absorption detection at 276 nm and a flow rate of 0.8 mL/min was
used. The retention times of L-tyrosine and 3-nitrotyrosine peaks
were determined using commercial standards. All determinations
were repeated at least three times, and the average of these
determinations is reported.
Fig. 1. Alanosine and MNP structures.

P. Sanchez-Cruz, A.E. Alegria / Journal of Photochemistry and Photobiology A: Chemistry 330 (2016) 79–85
81
2.2.4. Superoxide production
Superoxide production was measured using the SOD-inhibit-
able ferricytochrome c reduction method, as described elsewhere
100
80
60
40
20
0
[26,27]. Air-saturated solutions containing 0 or 1 mM HX, 10
mM
AlPcS4 and 50 M ferricytochrome C were irradiated at 675 nm for
m
10 min followed by measuring the solution absorbance at 550 nm.
The latter was performed in the presence and absence of 100 U/mL
of SOD. Differences in absorbances of irradiated solutions in the
presence and absence of SOD correspond to the SOD-inhibitable
absorbances. The latter are proportional to the superoxide
concentration produced.
3. Results and discussion
0
5
10
15
time / min
3.1. Photoirradiation of alanosine-containing solutions
Photoirradiation, at 675 nm, of a N2-saturated sample, con-
Fig. 3. Increase in the lowest-field EPR peak intensity of carboxy-PTI with
irradiation time at 675 nm of a N2-saturated sample containing 500 M carboxy-
PTIO, 500 M alanosine, AlPcS4 (A = 1) and 50 mM phosphate buffer (pH = 7.4).
m
m
taining 4 mM carboxy-PTIO, 10 mM AlPcS4, 500 mM alanosine and
50 mM phosphate buffer (pH 7.4) produces the NO-derived
carboxy-PTI EPR spectrum, Fig. 2 b), indicating the photosensitized
production of NO. Carboxy-PTIO and carboxy-PTI spectra are
diaziridinyl-1,4-benzoquinone enhances the photosensitized oxi-
dation of HX by, presumably, inhibiting back electron transfer from
the reduced dye to the HX cation [30]. Thus we tested if UBQ-0
presents a similar behavior in the present system by enhancing
characterized by splitting constants aN (2Ns) = 8.10 G and
g
value = 2.0062 for carboxy-PTIO and aN (1N) = 9.8 G, aN
(1N) = 4.4 G and g value = 2.0059 for carboxy-PTI, in agreement
with reported values [28,29]. The lowest field EPR peak of carboxy-
PTI increases in intensity as a function of irradiation time, Fig. 3. If
an identical sample is saturated with air, a smaller intensity of the
lowest-field carboxy-PTI EPR peak is detected as compared to that
corresponding to the N2-saturated sample, after 15 min of
irradiation, Fig. 2 d) vs b). This indicates that oxygen is competing
against alanosine in reacting with the dye excited state. In a
previous work we reported that the quinone 2,5-dichloro-
alanosine photosensitized oxidation. In fact, addition of 500 mM
UBQ-0 to an air-saturated solution of alanosine with the same
composition as described above increases the intensity ratio of the
lowest-field EPR peak of carboxy-PTI to that of the lowest-field EPR
peak of carboxy-PTIO by a factor of 2, Fig. 2 d) vs e). An increase by a
factor of ca. 3.5 in the same ratio of peak intensities is detected,
upon 500 mM UBQ-0 addition, in N2-saturated solutions, Fig. 2 b)
vs c). The larger increase in carboxy-PTI production upon UBQ-0
addition under N2- vs air-saturated conditions should be due to the
absence of oxygen competition for both the excited and reduced
b)
d)
a)
c)
f)
e)
Fig. 2. EPR spectra obtained after 15 min irradiation at 675 nm of samples containing 0 or 1 mM alanosine, 0 or 10
buffer (pH 7.4). Specific additional conditions of each sample are: (a) 0 mM alanosine, 10 M AlPcS4, N2-saturated; (b) 1 mM alanosine, 10
M UBQ-0; (d) 1 mM alanosine, 10 M AlPcS4, air-saturated sample; (e) 1 mM alanosine, 10
M AlPcS4, N2-saturated sample. Dotted-line spectrum in (e) corresponds to a spectral simulation and optimization
m
M AlPcS4, 500
m
M carboxy-PTIO in 50 mM phosphate
M AlPcS4, N2-saturated sample;
M AlPcS4, air-
m
m
(c) 1 mM alanosine, 10
saturated sample + 500
m
m
M AlPcS4, N2-saturated sample + 500
M UBQ-0; (f) 1 mM alanosine, 0
m
m
m
m
using WINSIM with parameters stated in Results and Discussion [34].

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P. Sanchez-Cruz, A.E. Alegria / Journal of Photochemistry and Photobiology A: Chemistry 330 (2016) 79–85
200
175
150
125
100
75
(a)
1 mM alanosine
3 mM MNP
50
25
0
0
25
50
75
100
125
Time/s
Fig. 4. NO traces after illumination at 670 nm of N2-saturated samples containing
1 mM alanosine or 3 mM MNP in the presence of 10 mM AlPcS4 and 50 mM
(b)
(c)
phosphate buffer (pH 7.4) at 37 ꢄC. Arrow indicates the instance illumination is
started.
dye in N2-saturated solutions. No carboxy-PTI is observed in the
absence of alanosine or dye, Fig. 2 a) and f). The observations stated
above suggest that a photosensitized oxidation of alanosine is
occurring since: (a) a similar behavior, i.e. quinone-induced
enhancement of the anaerobic photosensitized oxidation of HX
by AlPcS4 was observed previously [30] (b) enzymatic oxidation of
carbon-bound diazenium diolates produces NO [17] and (c) the
UBQ-0 semiquinone is detected.
Further evidence for the photosensitized production of NO was
obtained by the photoirradiation of a solution containing 1 mM
alanosine, 10 mM AlPcS4 and 50 mM phosphate buffer (pH 7.4)
under air- or N2-saturated conditions at the NO electrode chamber,
Fig. 4. Again, rates of NO formation are smaller in air- as compared
to N2-saturated conditions, Table 1. Furthermore, addition of
500 mM UBQ-0 to air-saturated or N2-saturated solutions increases
the rate of NO formation by ca. 12.3 and ca. 3.3, respectively,
Table 1. Evidence that electrons are being provided to UBQ-0, via
the reduced species of the dye, by alanosine oxidation, is the
appearance of the semiquinone of UBQ-0 in N2-saturated solutions
and only when both dye and alanosine are present in the irradiated
sample, Fig. 5.
(d)
Fig. 5. Epr spectra corresponding to irradiated N2-saturated solutions at 670 nm
Scheme 1 depicts a possible mechanism supported by these
observations. Light absorbtion by the dye generates the excited
singlet state of the dye which rapidly undergoes intersystem
crossing to the dye triplet state (Dye*). In the presence of oxygen
containing 50 mM phosphate buffer (pH 7.4), (a) 10
500 M UBQ-0, (c) 10 M AlPcS4, 0 mM alanosine and 500
AlPcS4, 1 mM alanosine and 500 M UBQ-0. Spectrum (b) is a spectral simulation
and optimization using WINSIM. Hyperfine coupling constants are aH(3Hs) = 2.44
G, aH(1H) = 1.95 G and aH(6Hs) = 0.03 G, which agree with previously reported
values [35].
m
M AlPcS4,1 mM alanosine and
m
m
m
M UBQ-0, (d) 0
mM
m
singlet oxygen (1O2, Type II pathway) and the superoxide ion (O2ꢀꢁ
,
Type I pathway) are generated. Another Type I pathway, which
could occur exclusively in the absence of oxygen, is the oxidation of
alanosine by the excited dye (reaction [4]). UBQ-0 will accept
electrons from the reduced dye reaction [5] followed by semi-
quinone disproportionation (reaction [6]), thus inhibiting back-
electron transfer (reverse of reaction [4]). Since the UBQ-0
concentration used (500 mM) should be much higher than the
steady state concentrations of both the intermediate species R-Dye
and alanosineꢀ+, a high probability for reaction [5] to occur, in
competition against reverse of reaction [4], is expected. Thus,
UBQ-0 reduction by the reduced dye could explain while UBQ-0
Table 1
Initial rates of NO formation, RNO, under several conditions, in irradiated samples at 675 nm containing 1.0 mM alanosine or 3.0 mM MNP, 10
buffer (pH 7.4) and the stated conditions. Errors are the standard deviations of the average of 3 determinations.
m
M AlPcS4, 50 mM phosphate
a
NO source
alanosine
N2-saturated
X
air-saturated
RNO (
m
M/s)
RNO+UBQ/RNO-UBQ
2.03 ꢃ 0.03
6.8 ꢃ 0.2
3.3
12.3
1.2
+500
m
M UBQ-0
X
0.12 ꢃ 0.03
1.47 ꢃ 0.04
2.4 ꢃ 0.2
+500
m
M UBQ-0
M UBQ-0
MNP
X
+500
m
M UBQ-0
2.8 ꢃ 0.1
X
+500
0.51 ꢃ 0.05
0.67 ꢃ 0.04
1.3
m
a
Ratio of RNO in the presence of UBQ-0 to that in its absence.

P. Sanchez-Cruz, A.E. Alegria / Journal of Photochemistry and Photobiology A: Chemistry 330 (2016) 79–85
83
1.6
1.5
1.4
1.3
1.2
0.1
0.2
0.3
[alanosine]-1 (mM-1)
Fig. 6. Determination of k4 for alanosine. Air saturated samples containing 10 mM
AlPcS4, alanosine in 50 mM phosphate buffer (pH 7.4) were irradiated at 670 nm in
the NO electrode chamber and initial NO rates were determined from the NO traces.
Table 2
Amounts of 3-nitrotyrosine obtained after photolysis at 675 nm of air-saturated
samples containing AlPcS4 (A = 1 at 675 nm), alanosine or MNP, 100 mM L-tyrosine
and 50 mM phosphate buffer (pH 7.4). Errors are the standard deviations of the
average of 3 determinations.
Scheme 1. Proposed mechanism for the photosensitized oxidation of alanosine and
its enhancement by UBQ-0, where H2UBQ-0 is the ubiquinone hydroquinone. The
dye is a tetraanionic species. The species R-Dye and O-Dye represents reduced and
oxidized species of the dye, respectively.
NO source
Irradiation time/min
[HX]/mM
[3-nitrotyrosine]/mM
2 mM alanosine
10
15
20
10
15
25
0
0
0
1
1
1
5 ꢃ 1
11 ꢃ 2
16 ꢃ 3
0
2 mM MNP
0
0.5 ꢃ 0.1
increases NO production in the case of alanosine-containing
samples. However, while the experimental observations plausibly
support the alanosine photosensitized oxidation, these are not
enough to rule out other possibilities. Further work regarding the
identification of other intermediates and products will aid in
establishing a more conclusive pathway.
3.3. Photoirradiation of MNP-containing solutions
The photosensitized release of NO by MNP solutions in the
presence of AlPcS4 has been reported previously [20] and
reproduced here, Fig. 4. Energy transfer from the triplet state of
AlPcS4 to the triplet state of MNP was postulated to lead to the
enhanced homolytic decomposition of MNP to generate NO and
the tert-butyl radical. Since MNP photosensitized oxidation is not
the pathway for NO release from MNP, UBQ-0 addition to MNP-
containing solutions has no such dramatic effect on the rate or
extent of the photosensitized NO release from MNP, Table 1. No 3-
nitrotyrosine formation was detected in air-saturated samples
containing MNP and AlPcS4, even after 25 min of irradiation. In a
previous work, we observed that a sacrificial electron donor such
as HX enhances the AlPcS4-photosensitized reduction of an
electron acceptor such as an alkylating quinone, via HX photo-
sensitized oxidation [30]. In another work we also reported that
xanthine (the 2-electron oxidation product of HX) enhances the
AlPcS4 photosensitized reduction of oxygen [33]. Thus, we
hypothesized in the present work that addition of HX to an air-
saturated MNP-containing solution should enhance oxygen
reduction with the consequent production of superoxide and,
therefore, peroxynitrite should be detected. In fact, if HX is added
to MNP + AlPcS4 solutions, 3-nitrotyrosine is detected after 25 min
of irradiation although in much smaller amounts than that
detected for alanosine + AlPcS4 solutions at an even shorter period
of irradiation, and with identical MNP and dye concentrations as
those used in alanosine-containing solutions, Table 2. That HX
enhances the photosensitized production of superoxide is proven
by our measurement of the SOD-inhibitable reduction of
ferricytochrome C. Addition of 1.0 mM HX to an air-saturated
The bimolecular rate constant for the reaction of alanosine with
the excited sensitizer (k4, see Scheme 1) is a measure of the ability
of the sensitizer to produce NO from alanosine and can be
determined as described previously by Singh et al. [20], assuming a
competition between alanosine and O2 in colliding with the
excited dye in air-saturated solutions. If the initial rate of NO
formation in air-saturated solutions at a given alanosine concen-
tration (RNO) is measured and compared to the limiting initial rate
at saturating alanosine concentration (RNO)max, the following
equation is obeyed:
1
kO ½O2ꢅ
1
2
¼
þ
ð9Þ
RNO k4½alanosineꢅðRNOmax
Þ
RNOmax
where kO2 and k4 are the bimolecular rate constants between
the excited state of AlPcS4 and 02 and alanosine, respectively. The
rate constant, kO2, when the dye is AlPcS4, has been reported as
1.8 ꢂ 109 Mꢁ1 sꢁ1 [31]. Thus, the bimolecular rate constant k4 can
be determined from the slope of the plot of 1/RNO vs 1/[alanosine],
Fig. 6. From the slope and intercept of that graph, the concentration
of oxygen and kO2, a value of k4 = (4.2 ꢃ 0.3) x 108 Mꢁ1 sꢁ1 is
obtained.
3.2. Photosensitized peroxynitrite production in alanosine-containing
solutions
Photoirradiation of an air-saturated sample containing 10
mM
AlPcS4, 2.0 mM alanosine, 100 M tyrosine and 50 mM phosphate
m
buffer (pH 7.4) produces 3-nitrotyrosine, as identified by HPLC
using a commercial standard, Table 2. The latter is indirect
evidence for peroxynitrite formation [32]. Thus, the reaction of the
photosensitized generation of superoxide (O2ꢀꢁ) with NO produces
the powerful oxidant and nitrating agent, peroxynitrite (ONOOꢁ).
solution containing 10 mM AlPcS4 and 50 mM ferricytochrome C in
50 mM phosphate buffer (pH 7.4), which was irradiated for 10 min
at 670 nm, produces an increase by a factor of 2.9 (0.058 ꢃ 0.002 vs
0.020 ꢃ 0.001) in the SOD-inhibitable absorbance at 550 nm. Thus,

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P. Sanchez-Cruz, A.E. Alegria / Journal of Photochemistry and Photobiology A: Chemistry 330 (2016) 79–85
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HX is providing the electrons for the increase in superoxide
production, and thus in peroxynitrite formation. Scheme
2
accounts for this observation. Reaction [10] in this Scheme has
been previously reported by Singh et al. [20] as the reaction
responsible for NO production. Reactions [11,12,8], account for HX
photosensitized oxidation, oxygen reduction and peroxynitrite
formation, respectively.
The fact that alanosine requires no additional sacrificial
electron donor to produce peroxynitrite, while MNP does, further
supports the idea that NO is released from alanosine through the
alanosine photosensitized oxidation pathway. In contrast to
alanosine, addition of UBQ-0 to MNP-containing solutions has
no significant effect on the extent of the photosensitized NO
production, since the mechanism of NO release by MNP is not
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4. Summary
The photosensitized production of NO from alanosine occurs
under aerobic and anaerobic conditions. While the presence of
UBQ-0 increases the rate of photosensitized NO production by
alanosine, with the consequent production of the UBQ-0 semi-
quinone under anaerobic conditions, it has little influence on the
rate of photosensitized production of NO by MNP. These
observations suggest that NO is produced by the photosensitized
oxidation of alanosine. In air-saturated solutions, AlPcS4 photo-
sensitize the production of peroxynitrite from alanosine while the
photosensitized production of peroxynitrite from MNP requires
the presence of HX as sacrificial electron donor. This work
demonstrates that a carbon-bound diazenium diolate such as
alanosine can be photosensitized to produce both NO and
peroxynitrite. The photosensitized production of NO could be
used in photodynamic therapies of malignancies where NO or
peroxynitrite are used as toxic agents and where nanoparticle
carriers, containing both the NO source and the photosensitizer,
are transported to the desired tissue.
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The authors are grateful to the Puerto Rico Science and
Technology Trust for the support of this work (grant # 2015-
00046) and to the NCI Developmental Therapeutics Program
(http://dtp.cancer.gov) for providing alanosine samples.

P. Sanchez-Cruz, A.E. Alegria / Journal of Photochemistry and Photobiology A: Chemistry 330 (2016) 79–85
85
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