Photosensitized oxidation of hypoxanthine and xanthine by aluminum phthalocyanine tetrasulfonate. Role of the alkylating quinone 2,5-dichloro-diaziridinyl-1,4-benzoquinone

Article abstract of DOI:10.1111/j.1751-1097.2008.00389.x

Photoirradiation of nitrogen-saturated aqueous solutions containing aluminum phthalocyanine tetrasulfonate (AlPcS₄) at 675 nm in the presence of 2,5-dichloro-diaziridinyl-1,4-benzoquinone (AZDClQ) and hypoxanthine (HX) produces the oxidized HX

Full text of DOI:10.1111/j.1751-1097.2008.00389.x

Photochemistry and Photobiology, 2008, 84: 1583–1588
Photosensitized Oxidation of Hypoxanthine and Xanthine by
Aluminum Phthalocyanine Tetrasulfonate. Role of the Alkylating
Quinone 2,5-Dichloro-diaziridinyl-1,4-benzoquinone
Antonio E. Alegria*, Yaritza Inostroza and Ajay Kumar
Department of Chemistry, University of Puerto Rico at Humacao, Humacao, Puerto Rico
Received 7 September 2007, accepted 23 April 2008, DOI: 10.1111/j.1751-1097.2008.00389x
anoxic ⁄ hypoxic cells. If the reducible substrate is a DNA-
ABSTRACT
alkylating quinone, which is activated by reduction, DNA
alkylation should be expected, with the consequent cell death,
if repair of the DNA damage is insufficient.
Photoirradiation of nitrogen-saturated aqueous solutions con-
taining aluminum phthalocyanine tetrasulfonate (AlPcS ) at
4
6
75 nm in the presence of 2,5-dichloro-diaziridinyl-1,4-benzo-
Diaziridinylbenzoquinones are alkylating agents by means
of their aziridine groups (9,10). These compounds have
significant activity against many tumor models and have been
in clinical use for several years (10). They contain a quinone
moiety that can be reduced and an aziridinyl group that can
form covalent bonds with a variety of cellular components,
including DNA. All aziridinyl compounds can be activated to
alkylate DNA by protonation of the aziridine groups followed
by nucleophilic attack. Indeed, it was demonstrated several
years ago that simple aziridinylbenzoquinones in aqueous
solution can cross-link DNA in the absence of reduction and
this process is pH dependent (11). The quinones that can
undergo bioreduction have an added advantage in many cases:
although the pK for the aziridine moiety is around 3–4, the pK
increases to around 5–6 when the quinone is reduced, and
hence the aziridine should more readily react with DNA bases
quinone (AZDClQ) and hypoxanthine (HX) produces the
oxidized HX derivatives, xanthine (X) and uric acid (UA).
Concentrations of the AZDClQ semiquinone, X and UA
increase at the expense of HX with an increase in irradiation
time. Almost negligible decomposition of HX, as well as very low
amounts of X, are detected if photolysis occurs under identical
conditions but in the absence of AZDClQ. Addition of calf-
thymus DNA produces quinone-DNA covalent adducts after
photolysis of anaerobic samples containing quinone, DNA and
4
AlPcS , in the presence or absence of HX and at pH 5.5.
However, larger amounts of quinone-DNA adducts are detected
if HX is present. The results presented here could have
applications in the photodynamic treatment of hypoxic tissues
such as solid tumors, under conditions of high HX concentration,
where Type-I pathways could be more important than singlet
oxygen generation.
(
(
(
12). Both the two-electron reduced species, hydroquinone
•
)
QH
14), have been proposed as the DNA-alkylating species.
2
) (13) and the semiquinone (Q ) of aziridinyl quinones
INTRODUCTION
Bioreductively activated drugs such as these are of potential
use against solid tumors containing hypoxic regions (15). In a
previous study, we found that PDT-type dyes under anaerobic
conditions photosensitize the reduction of alkylating quinones
with the subsequent binding of the reduced quinones to calf-
thymus DNA (16). Evidence suggested that, in the presence of
DNA, reducing equivalents were transferred from DNA to the
alkylating quinone.
Instead of DNA photoxidation by PDT dyes, sacrificial
electron donors could be used to improve the photosensitized
reduction of alkylating quinones. Sacrificial electron donors
are compounds that donate electrons to excited-state dyes.
Some of the biomedically relevant electron donors are man-
nitol, glucose, sucrose, maltose and amino acids such as
tryptophan, tyrosine, histidine, methionine, serine (17) and
cysteine (18). Other potential sacrificial electron donors are
hypoxanthine (HX) and xanthine (X). HX and X are sub-
strates of xanthine oxidase (XO). These purines are enzymat-
ically oxidized to the final product, uric acid (UA), by XO, if a
suitable reducible substrate, such as oxygen, is present (19).
Promising antitumor activity has been obtained in mice after
administration of a polythyleneglycol-bound XO (but not free
Photodynamic therapy (PDT) is a cancer treatment that uses a
combination of red laser light, a photosensitizing agent, and
molecular oxygen to produce the therapeutic effect (1,2).
Porphyrins, phthalocyanines, chlorins and other dyes are
currently being used in photodynamic treatment of tumors due
to their large absorption coefficients in the 500–800 nm range.
In the presence of dioxygen, these photosensitize the produc-
tion of singlet oxygen and superoxide (1,2). Singlet oxygen
production, the so-called Type-II pathway, is claimed to be the
most important process that kills tumor cells (1,2). However,
Type-I pathways, those involving photoreduction or photo-
oxidation of substrates, have also been proposed as photocy-
totoxic events in PDT, especially in hypoxic environments (3).
Solid tumors are often hypoxic (4). Therefore, direct killing
of the hypoxic cells by singlet oxygen is very limited (2,5).
However, as red–light-absorbing dyes also photoreduce oxy-
gen (6–8), these should also photoreduce molecules having
nearly equal or more positive redox potentials than oxygen in
*
ꢀ
Corresponding author email: antonio.alegria1@upr.edu (Antonio E. Alegria)
2008 The Authors. JournalCompilation. The AmericanSocietyofPhotobiology 0031-8655/08
1
583

1584 Antonio E. Alegria et al.
XO) followed by HX injection (20). Also, purine catabolites,
including HX, accumulate in solid tumors that are exposed to
hyperthermia (21). Thus, photolysis of PDT dyes in the
presence of both diaziridinylquinone and HX, or in combina-
tion with hyperthermia, could enhance quinone reduction
and macromolecule alkylation and thus improve PDT of solid
tumors.
In this work, we conduct a proof-of-principle test of a
mechanism whereby PDT could damage biomolecular targets
under hypoxic conditions. Specifically, we demonstrate that
A Shimadzu 10A analytical HPLC system, equipped with a Shimadzu
SPD-10 multiwavelength absorbance detector, was used at a wave-
length of 267 nm. Standard curves were prepared with HX, X and UA
solutions and used to obtain the concentrations of those species in
irradiated samples.
Samples of HX, X and UA of the irradiated solutions were also
collected from the HPLC analysis peaks and submitted to electrospray
ionization mass spectrometry (ESI-MS). These were performed on a
Micromass Quattro Micro API triple quadrupole mass spectrometer
with an ESI source (Micromass, Inc., Manchester, UK). Samples were
injected into the MS by direct infusion through capillary tubing at a
)
1
flow rate of 4 lL min . The full scan ESI conditions for the
monitored compounds were as follows: negative ionization mode,
capillary voltage (3.07 kV), cone voltage (20.00 V), extractor voltage
4
aluminum phthalocyanine tetrasulfonate (AlPcS ), under
nitrogen-saturated conditions (to simulate solid tumor regions
where oxygen participation is limited) photooxidizes HX to
produce X and X to produce UA in the presence of AZDClQ
but not in its absence. In addition, the presence of HX
increases the photosensitized formation of aziridinylquinone-
DNA covalent adducts under these conditions, thus suggesting
a Type-I process that could be exploited in the PDT of
solid tumors.
(
desolvation gas flow of 400 L h
2 V), source temperature (90ꢁC), desolvation temperature (300ꢁC) and
)1
.
Photosensitized semiquinone formation. Samples with the same
composition as that described above were photolyzed, at pH 7.4, for
increasing periods of time followed by transference of these to
deaerated EPR quartz flat cells. EPR spectra were then recorded on
a Bruker ER-200D spectrometer at 100 kHz magnetic field modula-
•)
tion. Semiquinone (Q ) concentrations were obtained by comparing
the semiquinone overmodulated EPR double-integrated spectral area
with that corresponding to
pyrrolin-1-oxide spin standard.
a 3-carbamoyl-2,2,5,5-tetramethyl-3-
Quinone covalent binding to DNA. The procedure used in this
determination was adopted from that described by Lusthof et al. (24)
and used previously by us (16,25). Solutions of DNA, dyes and
quinone were prepared, some were photolyzed and the samples were
MATERIALS AND METHODS
4
Materials. The dye, AlPcS , was obtained from Frontier Scientific
and used as received. AZDClQ, HX, X and UA were purchased
from Sigma-Aldrich Chemicals and used as received. The quinone
then left standing for 20 h at room temperature under an N
2
atmosphere and shielded from light by aluminum foil. The nonphoto-
lyzed samples served as matched controls for the photolysis. Then,
quinone-bound and -free DNA were separated from unbound quinone
on a Sephadex spun exclusion column (26). The DNA fractions were
analyzed by absorption spectroscopy. The presence of a quinone
absorption band at wavelengths above 300 nm was taken as evidence
of DNA-bound quinone. For example, the DNA-2,5-bis(1-aziridinyl)-
2,5-dichloro-diaziridinyl-1,4-benzoquinone was either purchased from
Sigma-Aldrich Chemicals or synthesized and characterized, starting
from the tetrachloro quinone, as reported elsewhere (22). Calf-
thymus DNA and Sephadex G-25 (superfine, DNA grade), were
purchased from Sigma-Aldrich Chemicals. All other chemicals were
of the highest purity commercially available and were used without
further purification. Aqueous DNA stock solutions were freshly
prepared in water each day and their concentration determined from
the absorbance at 260 nm and using a molar absorption coefficient
1
3
,4-benzoquinone adduct has an absorption maximum wavelength at
43 nm (24). For each photolyzed sample, Sephadex spun exclusion
chromatography was continued until no, or a small constant value of,
quinone absorption was detected in the matched but nonphotolyzed
control solution. Samples and controls were then extracted with an
equal volume of chloroform and then with an equal volume of diethyl
ether, followed by DNA precipitation and redissolution in water, as
described elsewhere (27). Samples and controls treated in this manner
were then submitted to absorption spectroscopic analysis. Sample pH
values were measured before and after photolysis and before Sephadex
extraction and were found to be constant. The molar ratio of
covalently bound quinone to DNA bases was estimated from the
quinone maximum absorbance in the 340–360 nm region, assuming
the same molar absorption coefficient as that of the parent quinone,
and DNA absorbance at 260 nm.
3
)1
)1
of 6.6 · 10
M
cm . Deionized and Chelex-treated water was used
in the preparation of all stock and sample solutions. All reagent
stock solutions were deaerated by flushing with water-saturated
nitrogen prior to mixing with other reagents. Oxygen was then
excluded from samples by keeping a positive pressure of N
the sample.
Sample photoirradiation. Nitrogen-saturated samples (1.00 mL)
2
gas in
)
1
containing 875 lM quinone in the presence or absence of 1 mg mL
calf-thymus DNA, in the presence or absence of 2 mM HX and a dye
concentration to produce an absorbance of 1, at the dye maximum
wavelength of 675 nm (6 lM), in 20 mM phosphate buffer at either pH
5.5 or 7.4, were photolyzed in quartz cuvettes of 1.00 cm light path
under continuous stirring. The pH value of 5.5 was selected to simulate
hypoxic tumor pH conditions (23). Wanting to keep the same
constituents at the two pH values and recognizing that phosphate
would not be an efficient buffer at pH 5.5, the pH value was measured
before and after irradiation and found to remain constant within ± 0.1
pH units. In addition, to avoid interference with the reactions,
phosphate was always used. Photoirradiation was allowed to occur for
periods of time ranging from 0 to 20 min at 675 ± 10 nm with a
radiant power of 8 mW. Samples were protected from external light
during and after preparation. Irradiations were performed using a 1000-
W xenon arc lamp coupled to a Schoeffel grating monochromator.
HX and X photooxidation. Samples were prepared and photolyzed
as indicated above followed by HPLC analysis of HX and the products
X and UA. HPLC analyses were performed using two Alltech
Platinum EPS C-18 (4.6 · 250 mm) columns and gradient elution
going from 100% ammonium acetate at pH 7.4 to 1:1 ammonium
acetate (pH 7.4):methanol in 15 min followed by 100% methanol. In
order to exclude oxygen from the eluant mixture, solvents were purged
with argon 30 min before injection and during the chromatographic
RESULTS AND DISCUSSION
HX and X photo-oxidation
When a nitrogen-saturated sample containing 2 mM HX,
8
4
75 lM AZDClQ and AlPcS (A = 1 at 675 nm) in 20 mM
phosphate buffer at pH 7.4 was photolyzed, the HX concen-
tration decreased while the X and UA concentrations
increased with irradiation time (Fig. 1). In addition to the
retention time, ESI-MS analyses of the collected HPLC peaks
show molecular masses, using negative ionization mode, of
[M-H]135.2, 151.4 and 167.3 corresponding to HX, X and
UA, respectively.
HX and X have been reported to be oxidized anaerobically
to yield X and UA, in the case of HX oxidation, and UA in
the case of X oxidation. For example, the anaerobic non-
photosensitized oxidation of HX or X by XO, which is
followed by UA detection, has been reported previously
)
1
analysis. The flow rate of elution was 1 mL min . The column was
washed daily with methanol for 1 h and then with water for 40 min
and stored in methanol:water (1:4, v ⁄ v). The column was re-
equilibrated to initial conditions for 60 min before injection.

Photochemistry and Photobiology, 2008, 84 1585
Figure 2. HX consumption after photolysis in samples containing
2.0 mM HX in 20 mM phosphate buffer in the presence or absence of
Figure 1. Dependence of HX, X and AU concentrations on photolysis
time and pH. The N -saturated sample contained 875 lM AZDClQ,
.0 mM HX, AlPcS (A = 1 at 675 nm) in 20 mM phosphate buffer at
pH 7.4 or pH 5.5 (only pH 5.5 is indicated). Data are presented as the
mean ± SEM of three samples for each condition.
875 lM AZDClQ, in the presence or absence of AlPcS (A = 1 at
4
2
)
1
2
4
675 nm) and in the presence or absence of 1 mg mL calf-thymus
DNA at pH 7.4 or 5.5.
(
(
see below), it disproportionates to form the parent quinone
Q) and hydroquinone (H Q), (Eq. 1).
(
28,29). The photosensitized oxidation of X to UA by
2
peroxydisulphate has also been reported previously (30).
Addition of a water molecule to the X cation radical to form
UA was proposed in that work and used in the present work
to propose a potential mechanism for X and UA production
ꢀ
ꢁ
þ 2Hþ ꢁ! Q þ QH2
2
Q
ð1Þ
(
see below). However, the photosensitized oxidation of X
Semiquinone formation
by Rose Bengal under aerobic conditions produces other
products instead of UA (30). The photosensitized aerobic
oxidations of HX, X and UA have also been reported by
others, although the products of those reactions were not
characterized (31,32).
As tumor environments are characterized by acidic pH
values (23), we also determined the yields of HX oxidation and
X formation at pH 5.5 (Fig. 1). The chromatographic peak
corresponding to UA was not quantified in this case, due to
severe overlap of this peak with others of unidentified products
of side reactions. As shown in Fig. 1, the yield of X, as well as
the extent of oxidation of HX, do not change, within
experimental error, upon going from pH 7.4 to 5.5.
Photolysis of a nitrogen-saturated sample containing 2 mM
HX, 875 lM AZDClQ and AlPcS4 (A = 1 at 675 nm) in
20 mM phosphate buffer at pH 7.4 produces the corresponding
semiquinone, AZDClQ , as detected by EPR spectrometry
(Fig. 3a). The semiquinone concentration shown in this figure
is the equilibrium concentration because its intensity did not
change appreciably after three or four scans of the spectrum.
Due to the large disproportionation constant of this semi-
•
)
1
7
quinone, i.e. 1.5 · 10
(Eq. 1) (25), the corresponding
hydroquinone should have also formed through the semiqui-
none disproportionation. As indicated above, the hydro-
quinone species is also postulated as DNA alkylator (12). As
the initial quinone concentrations are the same in nonirradi-
ated and irradiated samples, the fact that more semiquinone is
detected in irradiated samples is evidence that more reduced
quinone species are being produced in those samples. At the
more acidic pH of 5.5, a much smaller concentration of
semiquinone is generated due to increased semiquinone
disproportionation at acidic pH values (Eq. 1). Therefore,
only data at pH 7.4 are reported here. Much smaller
Furthermore, previous reports have shown that dyes and
visible light are able to photooxidize DNA under anoxia,
especially through guanine oxidation (33,34). However, addi-
tion of DNA to samples undergoing photolysis under anaer-
obic conditions at pH 5.5 containing AlPcS in the presence of
4
AZDClQ did not change the extent of HX oxidation within
experimental error (Fig. 2). The latter result could imply that
the rate of photo-oxidation of HX by AlPcS is much greater
4
than that of DNA base photo-oxidation under the conditions
of these samples. Thus, HX could be protecting DNA
from base photo-oxidation. Further work on this subject is
in progress.
semiquinone concentrations are obtained if either AlPcS or
4
HX is not included in the sample undergoing photolysis
(Fig. 3b). This basal semiquinone production could be
ascribed to autosensitization of the quinone (or direct dark
reduction by HX of the quinone), in the absence of AlPcS4,
activated by background room light, or from dye-photosen-
sitized auto-oxidation in the absence of HX. Thus, as expected
from the enhanced HX photo-oxidation in the presence of
quinone, more semiquinone is produced from electrons
provided by HX photo-oxidation than by other routes.
Interestingly, in the absence of quinone, HX oxidation is
strongly inhibited (Fig. 2), most probably due to back electron
transfer from the reduced dye to the HX cation radical.
Quinone enhancement of HX or X oxidation could be due to
inhibition of back electron transfer by semiquinone dispro-
•
)
portionation. Thus, once the semiquinone (Q ) is produced

1
586 Antonio E. Alegria et al.
•
)
Figure 3. Relative AZDClQ concentration produced as a function of
irradiation time of anaerobic samples containing 875 lM AZDClQ,
4
2.0 mM HX, AlPcS (A = 1 at 675 nm) and 20 mM phosphate buffer
at pH 7.4 and without HX or AlPcS
4
.
Quinone covalent binding to DNA
Absorption spectroscopic analysis of N
2
-saturated samples,
prepared and photolyzed as indicated above, show the presence
of covalentlyboundquinoneto DNA, atpH 5.5, ifAZDClQ and
4
AlPcS are both present in the photolyzed sample, and more if
HX is also present (Fig. 4). In a previous work, it was found that
no adduct was detected at pH 7.4 after photolysis of samples
Figure 4. (a) Absorption spectra highlighting the quinone absorption
band of N -saturated samples with the same composition as that stated
4
containing AlPcS , AZDClQ and DNA (15). Therefore, pH 7.4
2
was not used in the present work to detect covalent adducts. As
indicated above, this type of quinone is activated by both
protonation and reduction to become a DNA-alkylating agent.
The bound AZDClQ wavelength maximum (364 nm) is red-
shifted relative to that of the corresponding unreduced parent
quinone (339 nm). A similar red-shift in absorption wavelength
maximum has been reported for 2,5-bis(1-aziridinyl)-1,4-
benzoquinone upon reduction and alkylation of calf-thymus
DNA (24). The latter was ascribed to the formation of products
with open aziridine rings upon quinone reduction (24,35). If the
absorbance maximum of the bound quinone above 300 nm is
divided by the absorbance of DNA at 260 nm, the absorbance of
in Fig. 3a (but at pH 5.5) containing (+HX) or not containing ()HX)
hypoxanthine, which have been photolyzed for the indicated time
period followed by Sephadex spun filtration, solvent extraction and
DNA precipitation and redissolution. (b) Estimated relative fractions
of covalently bound quinone per unit amount of DNA (A ) and
Q
number of bound quinone molecules per 1000 DNA base pairs after
photolysis of samples with composition as described in Fig. 3a (pH
5
4
.5) and in the presence (+) or absence ()) of HX or AlPcS . These
fractions were determined after Sephadex spun filtration, solvent
extraction and DNA precipitation and redissolution of photolyzed
samples. Results shown are averages of three determinations.
irradiated for 5 min as compared with those irradiated for
30 min suggests that the decomposition of the quinone-DNA
covalent adduct can also be photosensitized. Thus, the irradi-
ation period needs to be optimized to maximize the net amount
of quinone-DNA adduct.
Q
quinone perunit DNA absorbance, A , is obtained. The number
of bound quinone molecules per 1000 bases can be estimated if
A is multiplied by the ratio of the extinction coefficient of DNA
Q
3
4
to that of the quinone (6.6 · 10 ⁄ 1.35 · 10 ) times 1000. The
latter is a measure of the relative abundance of bound quinone
among different sample conditions (Fig. 4b). The presence of
HX almost always induced the formation of a small amount of
covalent adducts in the absence of the dye, most probably due to
HX oxidation photosensitized by the quinone after background
room light absorption, or direct reduction of AZDClQ by HX
under dark conditions. This observation of DNA covalent
adducts, combined with the fact that bound quinone is observed
at pH 5.5 but not at pH 7.4 (16), strongly suggest that the bound
species is the aziridinyl quinone bound to DNA through its
opened aziridinyl group. The increased alkylation of DNA by
reduced aziridinylquinone at a lower pH than 7.4 indicates that
protonation of the aziridine ring is an essential step for
alkylation, which is in agreement with a high reactivity of
protonated aziridines toward nucleophilic species (36). As
binding of this type of quinone involves DNA cross-linking
Possible mechanism
A possible set of reactions involved in the system under study
here is shown in Scheme 1, where photo-oxidation of both HX
and X are postulated. This scheme is, of course, presented as a
hypothesis for further work as the proposed intermediates have
not been characterized yet in this system. That will be a matter
for a nearfuture study. Two electrons are transferred in both HX
and X oxidations (38). In contrast to monoxygenase systems,
XO generates rather than consumes reducing equivalents during
catalysis and utilizes water, as opposed to O , as the source of
2
oxygen for substrate hydroxylation (39). Swaraga et al. (30)
proposed that X can be oxidized in two stepwise reactions, by
two equivalents of SO4 , to produce UA. According to that
work, the nucleophilic attack of water at the C8 position in the X
cation radical occurs followed by further SO4 oxidation to
produce UA. The radical cation of another purine, i.e. guanine,
•)
•
)
(10,15,23), this type of damage should be difficult for a cell to
repair (37). The fact that more adducts are detected in samples

Photochemistry and Photobiology, 2008, 84 1587
HX+.
+
Dye
Dye + HX
[HX-OH]
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1
Scheme 1. Possible mechanism in accordance to observations reported
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1
1
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•
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•
are also shown in Scheme 1, where the [X-OH] structure is
taken from Ref. (30). Photoactivated water-derived hydroxyl-
ation has also been reported for other substrates. For example,
quinone photohydroxylation, in many cases, is postulated to
involve the reaction of the quinone triplet state, or cation radical
with water to produce the hydroxylating intermediate (41–45).
The photosensitized hydroxylation by water of 5,5-dimethyl-
pyrroline-N-oxide has also been reported (42,46). Once the
DNA adducts are formed by the reaction of DNA with the
quinone-reduced species, oxidation of the resulting adducts
when exposed to air during Sephadex spun exclusion produces
the observed quinone-DNA adducts.
In summary, the photosensitized oxidation, under anaero-
bic conditions, of HX and X to form X and UA is reported.
Observations in this work suggest that a PDT modality in
which a red–light-absorbing dye is photolyzed in the presence
of HX could result in larger yields of activated bioreductive
quinone in hypoxic regions of tumors, and thus an improve-
ment in the therapy.
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Acknowledgements—The authors express appreciation for grants P20
RR-016470 and S06-GM008216 from NIH for financial support of this
work and Professor Nancy L. Oleinick from Case Western Reserve
University and Professor Rafael Arce from University of Puerto Rico
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