Oxidation of 1-N2-etheno-2′-deoxyguanosine by singlet molecular oxygen results in 2′-deoxyguanosine: A pathway to remove exocyclic DNA damage?

Article abstract of DOI:10.1515/hsz-2017-0337

Exocyclic DNA adducts are considered as potential tools for the study of oxidative stress-related diseases, but an important aspect is their chemical reactivity towards oxidant species. We report here the oxidation of 1-N²-etheno-2′-deoxyguanosine (1,N²-?dGuo) by singlet molecular oxygen (¹O₂) generated by a non-ionic water-soluble endoperoxide [N,N′-di(2,3-dihydroxypropyl)-1,4-naphthalenedipropanamide endoperoxide (DHPNO₂)] and its corresponding oxygen isotopically labeled [¹⁸O]-[N,N′-di(2,3-dihydroxypropyl)-1,4- naphthalenedipropanamide endoperoxide (DHPN¹⁸O₂)], and by photosensitization with two different photosensitizers [methylene blue (MB) and Rose Bengal (RB)]. Products detection and characterization were achieved using high performance liquid chromatography (HPLC) coupled to ultraviolet and electrospray ionization (ESI) tandem mass spectrometry, and nuclear magnetic resonance (NMR) analyses. We found that dGuo is regenerated via reaction of ¹O₂ with the ?-linkage, and we propose a dioxetane as an intermediate, which cleaves and loses the aldehyde groups as formate residues, or alternatively, it generates a 1,2-ethanediol adduct. We also report herein the quenching rate constants of ¹O₂ by 1,N²-?dGuo and other etheno modified nucleosides. The rate constant (k_t) values obtained for etheno nucleosides are comparable to the k_t of dGuo. From these results, we suggest a possible role of ¹O₂ in the cleanup of etheno adducts by regenerating the normal base.

Full text of DOI:10.1515/hsz-2017-0337

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Biological Chemistry ’Just Accepted’ paper
ISSN (online) 1437-4315
DOI: 10.1515/hsz-2017-0337
Research Article
2
Oxidation of 1-N -etheno-2’-deoxyguanosine
by singlet molecular oxygen results
in 2’-deoxyguanosine: a pathway to remove
exocyclic DNA damage?
1
,
1
1
Glaucia Regina Martinez *, Hulyana Brum , Guilherme Lanzi Sassaki , Lauro Mera de
2
3
4
Souza , Ana Paula de Melo Loureiro , Marisa Helena Gennari de Medeiros and Paolo Di
4
,
Mascio *
1
Departamento de Bioquímica e Biologia Molecular – Setor de Ciências Biológicas – UFPR,
CP 19046 CEP 81531-980 Curitiba-PR, Brazil
2
Instituto de Pesquisa Pelé Pequeno Príncipe, Faculdades Pequeno Príncipe, Curitiba- PR,
Brazil
3
Departamento de Análises Clínicas e Toxicológicas, Faculdade de Ciências Farmacêuticas –
Universidade de São Paulo (USP), CEP 05508-000 São Paulo–SP, Brazil
4
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo (USP), CP
2
6077, CEP 05513-970 São Paulo-SP, Brazil
*
Corresponding author
e-mail: grmartinez@ufpr.br;
pdmascio@iq.usp.br
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1
Oxidation of 1-N -etheno-2’-deoxyguanosine by O
2
Abstract
Exocyclic DNA adducts are considered as potential tools for the study of oxidative stress-
related diseases, but an important aspect is their chemical reactivity towards oxidant species.
2
2
1
We report here the oxidation of 1,N -etheno-2’-deoxyguanosine (1,N -εdGuo) by O
generated by a non-ionic water-soluble endoperoxide (DHPNO ) and its corresponding
), and by photosensitization with two different
2
2
1
8
18
[
O]-oxygen isotopically labeled (DHPN O
2
photosensitizers (methylene blue and Rose Bengal). Products detection and characterization
were achieved using high performance liquid chromatography coupled to ultraviolet and
electrospray ionization tandem mass spectrometry, and Nuclear Magnetic Resonance
1
analyses. We found that dGuo is regenerated via reaction of O
2
with the ε-linkage, and we
propose a dioxetane as an intermediate, which cleaves and loses the aldehyde groups as
formate residues, or alternatively, it generates a 1,2-ethanediol adduct. We also report herein
1
2
the rate constant for O
2
to quench 1,N -εdGuo and other etheno modified nucleosides. The
rate constant (k ) values obtained for etheno nucleosides are comparable to the k
t
t
of dGuo.
1
From these results, we suggest a possible role of O in the cleanup of etheno adducts by
2
regenerating the normal base.
Keywords: alkylating damage; DNA adducts; etheno adducts; mass spectrometry;
oxidation; singlet oxygen.
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1
Oxidation of 1-N -etheno-2’-deoxyguanosine by O
2
List of abbreviations
1
2
O – singlet molecular oxygen
1
8
[
O] – oxygen isotopically labeled
2
2
1
1
3
8
,N -εdGuo – 1-N -etheno-2’-deoxyguanosine
6
6
,N -εdAdo -1,N -etheno-2’-deoxyadenosine
4
4
,N -εCyt – 3,N -ethenocytidine
-oxodGuo – 8-oxo-7,8-dihydro-2’-deoxyguanosine
CID – collision-induced dissociation
dGuo – 2’-deoxyguanosine
2
D O – deuterated water
DNA – deoxyribonucleic acid
DHPN – N,N’-di(2,3-dihydroxypropyl)-1,4- naphthalenedipropanamide
DHPNO2 – N,N’-di(2,3-dihydroxypropyl)-1,4- naphthalenedipropanamide endoperoxide
1
8
18
DHPN O – N,N’-di(2,3-dihydroxypropyl)-1,4- naphthalenedipropanamide endoperoxide isotopically labeled with
O
2
ESI – electrospray ionization
HPLC – high performance liquid chromatography
HSQC – heteronuclear single quantum correlation
k
k
k
q
r
t
– physical quenching constant
– chemical quenching constant
– quenching constant
MB – methylene blue
MIS – selected ion monitoring
MSn – multiple stage mass spectrometer
NL – neutral loss
NMR – Nuclear Magnetic Resonance
PDA – photodiode array detector
RB – Rose Bengal
UV – ultraviolet
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Oxidation of 1-N -etheno-2’-deoxyguanosine by O
2
Introduction
DNA adducts are products formed by the covalent reaction of electrophilic compounds with
nucleophilic sites in DNA. Carcinogenic compounds may have an intrinsic reactivity with
DNA or their reactivity may result from metabolic or photochemical activation of initially
non-reactive substances (Jeffrey and Straub, 1997).
Among the exocyclic DNA adducts, etheno-adducts (Figure 1) have been extensively studied
(
Medeiros, 2009). The interest to study these lesions began with the discovery that they are
induced by known occupational and environmental carcinogens, such as vinyl chloride, which
is metabolized to the potent alkylating agents chloroethylene oxide and chloroacetaldehyde by
microsomal monooxygenases (Barbin, 1998). The reaction of chloroacetaldehyde with
2
2
guanine produces two adducts: 1,N -ethenoguanine e 3,N -ethenoguanine (Figure 1), which
were first described by Sattsangi et al (1977). Another compound of interest is ethyl
carbamate (urethane), which is converted to vinyl carbamate, followed by oxidation to vinyl
carbamate epoxide, which is able to form the same adducts generated by the oxidation
products of vinyl chloride (Guengerich et al., 1991; Barbin, 2000). Furthermore, it was shown
that the formation of etheno-adducts can occur endogenously via the reaction of aldehydes
and epoxides resultant from the lipid peroxidation process with the DNA bases (Medeiros,
2
009, Guéraud, 2017).
Along the last two decades, many studies were dedicated to elucidate the sources, the
toxicological significance and consequences of these DNA lesions, and their repair pathways
(
Tudek et al., 2017). The exogenous factors or pathophysiological conditions that lead to their
increase, and whether these adducts could play a role in carcinogenesis or neurodegenerative
diseases have been addressed (Bartsch et al., 2011; Bartsch, 1999). The biological monitoring
of etheno-adducts could also provide information about the role of oxidative stress and lipid
peroxidation in pathophysiological processes and could contribute to the research of chemo
preventive agents (Bartsch and Nair, 2000; Dechakhamphu et al., 2010; Linhart et al., 2014).
Lipid peroxidation is also a source of peroxyl radicals that can lead to the formation of singlet
1
molecular oxygen ( O
2
) (Miyamoto et al., 2003a,b, 2006, 2014), an oxidizing species.
1
However, there is no information about the reactivity of O
2
towards DNA etheno-adducts.
The investigation of the chemical stability of the etheno-adducts is important to better
understand their biological roles.
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1
Oxidation of 1-N -etheno-2’-deoxyguanosine by O
2
The chemical lability of etheno-adducts is known from some studies (Yip and Tsou, 1973;
6
Basu et al., 1993; Speina et al., 2001, 2003; Barbati et al., 2010). The etheno-adduct 1,N -
6
6
etheno-adenine (1,N -εAde) in the free deoxynucleoside form (1,N -εdAdo) or incorporated in
oligodeoxynucleotides underwent depurination and gave ring-opened derivatives when kept in
neutral and alkaline conditions (Basu et al., 1993; Speina et al., 2001, 2003). One of the ring-
opened derivatives, 4-amino-5-(imidazol-2-yl)imidazole, was more mutagenic than the
6
parental 1,N -εAde when tested in Escherichia coli (Basu et al., 1993), and it was not repaired
by several DNA glycosylases (Speina et al., 2003). Instead, it was bypassed by DNA
polymerases, giving mutations, or depurinated, giving apurinic sites and subsequent DNA
2
strand breaks (Speina et al., 2003). The stability of the 1,N -etheno-2’-deoxyguanosine
2
(
1,N -εdGuo) adduct was tested under oxidation reactions with hydroxyl radical and sulfate
radical anion in vitro (Barbati et al., 2010). Regeneration of 2’-deoxyguanosine from
2
1
,N -εdGuo was observed as a result of the oxidative processes (Barbati et al., 2010).
1
Synthetic naphthalene endoperoxides are able to release pure O
2
and also the corresponding
1
8
[
O]-labeled upon gentle warming (Martinez et al., 2000). This approach was used before to
study the oxidation of 2’-deoxyguanosine or 8-oxo-7,8-dihidro-2’-deoxyguanosine free in
solution or in DNA (Duarte et al., 2000; Ravanat et al., 2000, 2001, 2006; Martinez et al.,
2
1
2
002, 2007). Here we present data on the oxidation of the etheno-adduct 1,N -εdGuo by O
2
,
characterizing the products formed by spectroscopic techniques. We took advantage of the
1
8
1
18
isotopic labeling using a chemical generator of [ O]-labeled O
2
(DHPN O
2
) (Martinez
towards
1
et al., 2000). Furthermore, we also report the total quenching constant of O
2
2
6
4
1
,N -εdGuo and other etheno-adducts, such as 1,N -εdAdo and 3,N -ethenocytidine
4
(
3,N -εCyt).
Results
2
1
2 2
The analysis of the reaction of 1,N -εdGuo with O generated by DHPNO shows three major
products (Figure 2a). Product A, with a retention time of 5.8 min, presents in the mass
+
spectrum a molecular ion [M + H] at m/z 326 and a fragment characteristic of the loss of
+
1
16 Da of 2'-deoxyribose, [BH + H] at m/z = 210 (Figure 2b). The product B, with retention
time of about 8.5 min, shows the mass spectrometric characteristics of dGuo, with the ion at
m/z 268 and the fragment at m/z 152 (Figure 2b). The product C with retention time of about
+
+
1
3 min, presents the ion signal of [M + H] at m/z 296, the sodium adduct [M + Na] at
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1
Oxidation of 1-N -etheno-2’-deoxyguanosine by O
2
+
m/z 318, and the fragment resultant from the break of the N-glycosidic bond, [BH + H] , at
+
2
m/z 180 (Figure 2b). The ion signal of [M + H] at m/z 292, corresponding to the 1,N -εdGuo,
was not detected after the reaction, indicating a high consumption under this condition.
The UV spectra of the three products were obtained by HPLC analysis with photodiode array
detector, which are shown in Figure 2c. Product A shows a spectrum very similar to B, which
in turn is identical to the spectrum of an authentic sample of dGuo (Figure 2d). The data
obtained show that the products formed have lost the characteristic structure of the etheno-
adduct, given the differences in the UV absorption spectra.
A reaction of photosensitization with Rose Bengal was performed also to confirm whether the
2
same product profile formed by DHPNO . As can be seen in Figure 3, the analysis
immediately after photosensitization clearly showed the formation of three major products, as
expected. Interestingly, after incubation of the crude reaction during a week in the dark at
room temperature, we observed an increase of product B (Figure 3).
Characterization of products A and C
The product A has a signal at m/z = 326 in the mass spectrum, this value might not match the
primary cleavage product of the dioxetane with two aldehyde groups because the signal would
+
be observed at m/z = 324. The compound A was observed at m/z 326.1113 [M+H] in the
HR-MS (Figure S4), consistent with a chemical composition of C12H16N5O6 (theoretical
m/z 326.10951), with a mass error of 5.5 ppm. A single fragment was observed at
m/z 210.0629, with a neutral loss (NL) of 116.0484 atomic mass units (a.m.u) consistent with
the loss of the 2-deoxyribosyl residue. However, the absence of other fragments hindered the
structure identification. To resolve this issue, the compound A was analyzed by multiple-stage
+
mass spectrometry, observed in MS1 at m/z 326 [M+H] . Using MS2 (CID), the fragment at
m/z 210 was produced (Figure 4), consistent with the loss of the deoxyribosyl residue. This
ion (m/z 210) was isolated and fragmented (MS3), producing fragments at m/z 192 (NL of
1
8 a.m.u., consistent with a loss of water), m/z 164 (NL 46 a.m.u., loss of CH2O2) and
m/z 152 (NL 58 a.m.u., loss of C2H2O2). The ion at m/z 192 was isolated and fragmented
MS4), giving rise to the fragment at m/z 164 (NL 28 a.m.u., loss of CO). The ion at
(
m/z 164 gave, in MS5, the fragment at m/z 135 (NL 29 a.m.u., loss of CH3N), exposing the
deaminated purine nucleobase (Figure 4).
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1
Oxidation of 1-N -etheno-2’-deoxyguanosine by O
2
2
The structural analysis of 1-N -εdGuo intermediates along the photooxidation was achieved
1
13
using heteronuclear H/ C-HSQC NMR spectroscopy. Following the literature and deduced
1
13
chemical shifts it was possible to assign the hydrogen and carbon resonances of H/ C atoms
of 1,2-di-OH-ethane, shown in Figure 5. As noted in the HSQC spectrum, the anomeric
signals were identified at δ 4.409/83.7 and δ 4.847/82.5 namely (a) and (b), respectively.
Other signals from N-2-deoxi-Ribf were also observed at δ 5.978/83.7 (84%) and δ 5.846/83.6
2
(
16%). The signals of guanine moiety from 1-N -εdGuo were present at δ 7.700/138.8, δ
7
.203/106.3 and δ 6.906/116.6 and oxidized at δ 7.548/137.7 and δ 7.5175/137.5.
2
The protons from ethane diol during the photooxidation of 1-N -εdGuo (a) at δ 5.409 and (b)
at δ 4.847 were selected individually spins for magnetic excitation, then the spectra were
collected with 20 ms and 100 ms. The proton signals represented by (b) was observed as the
closest to the selected peak (a), respectively, due to the vicinal scalar coupling. Owing to this
2
fact, it is possible to confirm the 1,2-dihydroxyethane-1-N -εdGuo occurrence in the reaction.
2
18
2
After incubation of 1,N -εdGuo with DHPN O , analyses were performed by HPLC with UV
detection and mass spectrometry. In Figure 6, we observe the chromatograms obtained by UV
detection at 260 nm and the chromatograms obtained for product C by selected ion
monitoring (MIS) when the analysis was performed in full scan mode, neutral loss of 116 and
daughters of 298. The product C had the incorporation of oxygen, because of an increase in
molecular weight due to the incorporation of an oxygen atom-18.
Total quenching constants determination
1
Total deactivation constants (k
t
) were determined from O
2
luminescence decay at 1270 nm
using deuterated water (D
2
O) as the solvent, figure S5 and table 1 show the traces and values
1
obtained for the different compounds. The experimental values for the mean lifetime of O
2
in
D
2
O were around 65 μs.
2
We determined the quenching constants of singlet oxygen by 1,N -εdGuo and other etheno
6
4
modified nucleosides (1,N -εdAdo and 3,N -εCyt) for comparison. The constants showed the
same order of magnitude obtained for dGuo, indicating that these products can be regarded as
1
targets of the reaction with O
2
.
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Oxidation of 1-N -etheno-2’-deoxyguanosine by O
2
Discussion
o
Sattsangi et al. (1977) studied the reaction of guanosine with chloroacetaldehyde at 37 C in a
pH range from 6.5 to 4.5. They reported that the relative rate of the reaction with guanosine
decreases with increasing acidity of the medium and is almost negligible at pH 4.5. The
2
formed product was characterized as 1,N -ethenoguanosine. In the present work, the reaction
o
of dGuo with chloroacetaldehyde was performed at 50 C in the pH range from 6.5 to 12. We
2
observed that at pH 8.0 the yield of 1,N -εdGuo is lesser than at pH 6.5. Also, at pH 12, there
is no formation of the product (Figure S1), probably by the instability of etheno adducts in
alkali. In addition, the chromatogram of Figure S1 shows the presence of many other
products, even at pH 6.5, which required further purification. After purification of the crude
extract reaction in the solid phase extraction C-18 cartridges, the fractions were pooled and
lyophilized. Only the samples eluted with methanol 10 and 20% showed the presence of the
product. The product was purified on a semi-preparative scale from fractions 10 and 20%
CH
3
OH obtained in the pre-purification step.
2
1
The analysis of the reaction of 1,N -εdGuo with O generated by DHPNO shows three major
2
2
products with spectral data that indicate cleavage of etheno linkage (Products A and C) and
regeneration of parental nucleoside dGuo (product B). The same pattern was observed to RB
photosensitization. We need to emphasize that under the conditions of RB-irradiation
1
employed here, only O
2
is generated because of the narrow wavelength from the green LED
employed (Allen, 1991). A reaction of photosensitization with methylene blue (MB) was
performed to verify whether the same product profile formed by DHPNO or RB was
2
obtained. As can be seen in Figure S2 and S3, the analysis immediately after
photosensitization clearly show the formation of three major products, as expected. The
HPLC analysis with detection by mass spectrometry showed the same products A, B and C
2
obtained previously with DHPNO and RB.
2
1
These results show that 1,N -εdGuo is susceptible to oxidation by O , whose mechanism
2
probably involves the attack on the double bond forming a dioxetane intermediate, which
after cleavage and loss of the aldehyde as a formyl group by the action of water affords the
product with mass spectrum signal at m/z = 296 (Product C), this product may suffer loss of
the second aldehyde group, yielding dGuo as final product (Scheme 1). Our results indicate
that product A has an ethane-1,2-diol group. The presence of a diol group was not an expected
product from the dioxetane decomposition. However, evidence obtained in this investigation
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1
Oxidation of 1-N -etheno-2’-deoxyguanosine by O
2
support this statement, since after losing the deoxyribose moiety, the precursor-fragment
m/z 210) undergoes a dehydration to produce the fragment at m/z 192, that occurs in
(
hydroxylated molecules. Additionally, the most of other fragments were produced by losing
oxygenated components, until the release of the central core of the nucleobase. These data
lead to the proposal of the diol structure, which was confirmed by NMR analyses. Besides
getting solid information about the chemical structure of product A, the mechanism for its
generation could not be totally clarified at this moment, but our data (See Figure 3) also
indicates that this compound also generates dGuo (Scheme 1).
1
2
2
In order to better establish the biological significance of O reaction towards 1,N -εdGuo, the
determination of the quenching constant is of fundamental importance. The deactivation of
1
2
O by substrates can occur by a physical or chemical mechanism. The physical pathway
involves the interaction of oxygen with a (deactivator) substrate that catalyzes its conversion
to the ground state without consuming oxygen or forming products. In the chemical
quenching, there is oxygen consumption and product formation. The total quenching constant
t q r t r q
(k ) is the sum of physical (k ) and chemical (k ) constants (k = k + k ). There are two main
physical mechanisms for deactivation: energy transfer and charge transfer. In both cases, it is
generally accepted the formation of a transient and reversible intermediate. According to the
excitation energy and the ionization potential of the deactivator, the intermediate can be
described as a covalent adduct (perepoxide) intermediate, or as an exciplex or as a charge
transfer complex (Sheu and Foote, 1995).
It has been reported in the literature the total deactivation constant of guanosine derivatives
with hydroxyl groups of the ribose protected by t-butyldimethylsilyl guanosine substituted at
3 2 6 5 3
position 8 with Br, CH , OCH C H , OCH and OH (8-oxodGuo) in different deuterated
solvents (dichloromethane, benzene, acetone, and acetonitrile). The results showed that k
t
increased about 5 to 15 times for all derivatives in polar solvents (acetonitrile in this case),
and this sensitivity to solvent polarity was more pronounced to derivatives with electron rich
substituents, indicating that the transition into this state is by polar interaction with partial
charge transfer (Sheu and Foote, 1995).
6
-1 -1
In our study, k determined for dGuo and 8-oxodGuo in D O were 5.39×10 M s and
t
2
6
-1 -1
5
8.7×10 M s , respectively. When compared with the values determined by Sheu and Foote
(
1995) for the same compounds but with the deoxyribose protected by t-butyldimethylsilyl in
6
-1 -1
6
-1 -1
acetonitrile the values obtained by those authors were 9.15×10 M s and 65.2×10 M s ,
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1
Oxidation of 1-N -etheno-2’-deoxyguanosine by O
2
respectively. The values obtained in this work for the dGuo and 8-oxodGuo are lower because
of the effect of solvent on the constant (water is more polar than acetonitrile), besides, the
hydroxyl groups of 2'-deoxyribose were not protected, which could contribute to the
1
deactivation of O
2
by the transfer of electronic excitation energy to vibrational energy levels
6
-1 -1
of the OH bond. Moreover, the value of 5.3×10 M s has been reported previously for dGuo
in an aqueous system (Lee et al., 1987), which is very close to that determined in the present
work.
t
Also, k for the physical deactivator sodium azide was determined and the value obtained
8
-1 -1
(
5.28×10 M s ) is in agreement with values previously reported in the literature that are in
8
-1 -1
the range of 3 to 6.9×10 M s (Lissi et al., 1993). Interestingly, other authors also tested
2
1
,N -εdGuo reactivity towards hydroxyl radical and one electron oxidation reactions, and they
2
also concluded that the exocyclic ring of 1,N -εdGuo is mainly affected by oxidative
processes leading to the regeneration of 2'-deoxyguanosine (Barbati et al., 2010). Our results
showed the same product by a different reactive oxygen species. In this way, the condition of
oxidative stress may contribute to the degradation of the etheno-adducts and regeneration of
the original guanine base, contributing to the maintenance of DNA or nucleotide pool.
Materials and methods
2
Preparation and purification of 1,N -εdGuo
As described by Sattsangi et al. (1977), the reaction of dGuo with chloroacetaldehyde
2
generates the product, 1,N -εdGuo (Reaction 1). Thus, incubations were done using dGuo
(
Sigma-Aldrich Brasil, Brazil, 4 mM) and chloroacetaldehyde (Sigma-Aldrich Brasil, Brazil,
0 mM) in phosphate buffer pH 6.5, 8.0 and 12 at 50° C for 5 days or at 37° C for 4 days.
6
O
N
O
N
N
N
N
N
pH 6.5
HN
N
O
+
+
HCl
+
H O
2
Cl
H N
N
2
H
dR
dR
(
Reaction 1) dR= 2-deoxyribose
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Pre-purification of the adduct was performed by C-18 solid phase extraction (Supelclean-1G-
ml, Sigma-Aldrich Brasil, Brazil). The C-18 cartridge was equilibrated with 12 ml of
6
methanol (Merck, Brazil) and then with 12 ml of water. A volume of 300 μL of the reaction
solution was applied to the cartridge and the products were eluted by the following sequence
of solvents: 1 ml of water, 3 ml of 2% CH
3
OH, 3 ml of 5% CH
3
OH, 6 ml of 10% CH
3
OH,
6
ml of 20% CH OH, 6 ml of 80% CH OH and 6 ml of 100% CH
3
3
3
OH. The fractions were
lyophilized for further purification by HPLC-UV (Shimadzu, Japan).
2
The adduct 1,N -εdGuo was purified by semi-preparative HPLC of the 10 and 20% CH
3
OH
fractions from the pre-purification step. The UV detector was set for detection at 225 nm. The
column used was a LC-18-Hypersil ODS (Thermo, 250×10 mm, particle size 10 μm) with a
flow rate of 4.5 ml/min. The separation was performed with a linear gradient starting with 5%
3 3
CH CN/95% water for 5 min, changing to 10% CH CN until 20 min, then to 60% in the next
5
minutes, staying at this condition until 30 min and returning to 5% CH CN until 35 min.
3
2
Incubation of 1,N -εdGuo with singlet molecular oxygen
2
Solutions were prepared in deuterated water (Sigma-Aldrich Brasil, Brazil) containing 1,N -
1
8
εdGuo (0.5 mM) and DHPNO
2
or DHPN O
2
(10 mM) or DHPN Δ (10 mM, obtained after
1
8
thermo decomposition of DHPNO
2
), and incubated at 37°C for 2.5 h. DHPNO or DHPN O
2 2
were prepared was previously reported (Martinez et al., 2000).
2
Photosensitization with methylene blue: a solution of 1,N -εdGuo (0.5 mM) and methylene
blue (Merck, Brazil, 20 μM) in deuterated water was irradiated with a 500W lamp (Philips,
Brazil) under oxygen bubbling for 1.5 h at 10°C in a thermostatically controlled water bath.
2
Photosensitization with Rose Bengal: a solution of 1,N -εdGuo (0.1 mM) and Rose Bengal
(
(
Sigma-Aldrich Brasil, Brazil, 200 μM) in water was irradiated for 115 min with green LEDs
2
Cromatek, Brazil, 526 nm) with an irradiance of 1.67 ± 0.13 mW/cm .
2
Analysis of the products generated by the incubation of 1,N -εdGuo with singlet
molecular oxygen
The products were analyzed using a Shimadzu HPLC equipment with a photodiode array
detector (PDA) programmed to detect the wavelength range of 200-370 nm. The column was
a LC-18 (250×4.6 mm, particle size 5 μm) from Supelco (Sigma-Aldrich Brasil, Brazil) with a
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flow rate of 1 ml/min. The separation was performed with a linear gradient starting with 5%
CH CN/95% water for 5 min, reaching 10% CH CN in 20 min, and 60% in 25 min, and
returning to 5% CH CN until 30 min.
3
3
3
The analyses by mass spectrometry (Quatro II, Micromass, Waters) were performed in the
+
positive electrospray ionization mode (ESI ) with the following specifications: drying gas
flow and nebulizer (both nitrogen) were 300 l/h and 30 l/h, source temperature: 120°C,
capillary potential, and HV electrode were 3.5 and 0.3 kV, respectively. The cone potential
was set at 30 V and spectra were acquired in the first analyzer in the range from 50 to
4
00 m/z.
Structural characterization of product A by mass spectrometry and nuclear
magnetic resonance spectroscopy measurements
Product A was isolated by HPLC, lyophilized, and analyzed by mass spectrometry with
electrospray ionization at atmospheric pressure ionization using a multiple stage mass
spectrometer (MSn), LTQ-XL (Thermo Fisher Scientific™). The MSn analysis was
developed in a Linear Ion Trap, by direct infusion of the sample, that was dissolved in water
and infused at 10 μl/min. Nitrogen was used for sample desolvation, at flow rates of 8 and 2
arbitrary units, in the sheath and auxiliary gas, respectively, with the source temperature held
o
+
at 300 C. Positive ions [M+H] were obtained applying 4.5 kV to the ion source, 40 V to the
capillary and 130 V to the tube lens. The analyte was fragmented by collision-induced
dissociation (CID) with 20 normalized collision energy in the MS2-MS4, in the MS5 the
energy was 25. Instrument calibration is routinely performed, using Pierce™ LTQ ESI
Positive Ion (Caffeine, MRFA, Ultramark-1621).
2
For NMR analysis, the reaction was carried out using 400 µl 1-N -ƐdGuo 2.28 mM, 7.3 µl
Rose bengal 5 mM and 504.7 µl D O totalizing 912 µl and samples loaded into a 5 mm tube.
2
The nuclear magnetic resonance spectra were obtained using a Bruker Avance III 14.1 T
spectrometer operating at 600 MHz and the data were acquired in an inverse 5-mm probe
1
head (TXI) at 303 K. 1D H-NMR were performed after 90 ° (p1=9.25 μs) pulse calibration.
Residual HDO was pre-saturated with a pulse programme zgpr (TOPSPIN 3.2, Bruker
Biospin, Rheinstetten, Germany) using a relaxation delay = 4.0 s, a number of time domain
points = 65,536 and acquisition time = 5.1 s to obtain a spectrum width of 6393 Hz, using
1
13
3
2 data points. H and C chemical shifts were determined by 2D NMR analyses using
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Oxidation of 1-N -etheno-2’-deoxyguanosine by O
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1
13
HSQC (hsqcedtgpsisp2.2), using 9014 Hz ( H) and 30184 Hz ( C) widths and a recycle delay
of 1.160 s. The spectra were recorded for quadrature detection in the indirect dimension, using
16 scans per series of 2048×256 data points with zero filling in F1 (2048) prior to Fourier
transformation. All signal assignments were performed using TopSpin program. 1D TOCSY
2
selective excitation of compound (A), 1,N -ethenodiol-2’-deoxyguanosine experiments were
carried out using a standard Bruker pulse program (selmlgp). A shaped pulse length of 80 ms
for selective excitation was used, followed by an MLEV-17. Selective pulses of different
mixing times varying from 20-100 ms using 256 scans were used.
Total quenching constants determination
1
Phenalenone was used as the O
2
photosensitizer and it was irradiated by a Nd:YaG laser at
3
55 nm. Monomol emission at 1270 nm was monitored with a special photocounting
apparatus equipped with a monochromator suitable for emissions in the near-infrared region
1
(
800–1400 nm) which enabled us to follow the kinetics of formation and decay of O
2
and to
determine its spectrum. The apparatus consists of a R5509 PMT photomultiplier tube from
Hamamatsu Photonics (Shizuoka, Japan), cooled to -80°C with liquid nitrogen in a S600
PHOTOCOOL, PC176TSCE005 cooler from Products for Research Inc. (Danvers, MA) to
reduce the dark current, and powered by a Model C3360 high voltage DC power supply from
Hamamatsu Photonics with the applied potential set to -1.5 kV. The light emitted from the
sample passed through a model M300 monochromator equipped with a Type G306R1U0
diffraction grating from Bentham Instruments Ltd (Reading, UK). The monochromator and
the acquisition of data were controlled by F900 software version 6.22 software from
Edinburgh Analytical Instruments (Livingston, UK). Experiments were conducted at room
temperature in a quartz cuvette under continuous stirring with a small magnetic bar.
1
The rate constant for the total (physical and chemical) quenching (k
t
) of O
2
was determined
for each compound by employing different concentrations and Stern-Volmer plots
Wilkinson, 1993). For comparison, the k of sodium azide, 2’-deoxyguanosine (dGuo) and
-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodGuo) were also determined in the same
conditions.
(
t
8
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Oxidation of 1-N -etheno-2’-deoxyguanosine by O
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Acknowledgments
Paolo Di Mascio thanks FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo,
No. 2012/12663-1, CEPID Redoxoma Paolo Di Mascio and Marisa H.G. Medeiros No.
2
013/07937-8), CNPq (Conselho Nacional para o Desenvolvimento Científico e Tecnológico,
No. 301307/2013-0, No. 159068/2014-2, Marisa H.G. Medeiros No. 301404/2016-0 and
Glaucia R. Martinez, No. 311629/2013-0), PRPUSP (Pro-Reitoria de Pesquisa da
Universidade de São Paulo, NAP Redoxoma No. 2011.1.9352.1.8), CAPES (Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior) and John Simon Guggenheim Memorial
Foundation (P.D.M. Fellowship) for financial support. G. R. Martinez, G.L. Sassaki, A. P. M.
Loureiro, M. H. G. Medeiros, and P. Di Mascio received fellowships from CNPq.
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References
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dependent mechanism for rose bengal-sensitized photoinhibition of red cell
acetylcholinesterase. Biochim. Biophys. Acta 1075, 42-49.
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Barbati, S., Bonnefoy, A., Botta, A., Chiron, S. (2010). Secondary oxidation of cyclic 1,N -
2
propano and 1,N -etheno-2'-deoxyguanosine DNA adducts. Consequences in oxidative
stress biomarker development. Chemosphere 80, 1081-1087.
Barbin, A. (1998). Formation of DNA etheno adducts in rodents and humans and their role in
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Barbin, A. (2000). Etheno-adduct-forming chemicals: from mutagenicity testing to tumor
mutation spectra. Mutat. Res. 462, 55-69.
Barstch, H., Nair, J. (2000). New DNA-based biomarkers for oxidative stress and cancer
chemoprevention studies. Eur. J. Cancer 36, 1229-1234.
Barstch, H., Nair, J. (2006). Chronic inflammation and oxidative stress in the genesis and
perpetuation of cancer: role of lipid peroxidation, DNA damage, and repair.
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Bartsch, H. (1999). In: Exocyclic DNA adducts in mutagenesis and carcinogenesis, Keynote
address: Exocyclic adducts as new risk markers for DNA damage in man, Singer, B.;
Bartsch, H., Eds. (Lyon, France: IARC Sci. Publ.), 150.
Bartsch, H., Arabi, K., Nair, J. (2011). Biomarkers for hazard identification in humans. J.
Environmental Health 10 (Suppl 1), S11.
Basu, A.K., Wood, M.L., Niedernhofer, L.J., Ramos, L.A., Essigmann, J.M. (1993).
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4
ethenoadenine,
3,N -ethenocytosine,
and
4-amino-5-(imidazol-2-yl)imidazole.
Biochemistry 32, 12793-12801.
Dechakhamphu, S., Pinlaor, S., Sitthithaworn, P., Nair, J., Bartsch, H., Yongvanit, P. (2010).
Lipid peroxidation and etheno DNA adducts in white blood cells of liver fluke-infected
patients: protection by plasma α-tocopherol and praziquantel. Cancer Epidemiol
Biomarkers Prev. 19, 310-318.
Duarte, V., Gasparutto, D., Yamaguchi, L.F., Ravanat, J-L., Martinez, G.R., Medeiros,
M.H.G., Di Mascio, P., Cadet, J. (2000). Oxaluric acid as the major product of singlet
oxygen-mediated oxidation of 8-oxo-7,8-dihydroguanine in DNA. J. Am. Chem. Soc.
1
22, 12622–12628.
6
El Ghissassi, F., Barbin, A., Nair, J., Bartsch, H. (1995). Formation of 1,N -ethenoadenine
4
and 3,N -ethenocytosine by lipid peroxidation products and nucleic acid bases, Chem.
Res. Toxicol. 8, 278-283.
Guengerich, F.P., Kim, D.H., Iwasaki, M. (1991). Role of human cytochrome P-450-IIE1in
the oxidation of many low-molecular-weight cancer suspects. Chem Res. Toxicol. 4,
1
68-179.
Jeffrey, A.M., Straub, M.I. (1997). In: Encyclopedia of Cancer, Carcinogen-DNA Adducts,
Academic Press, Vol. 1, pp. 207-219.
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1
Oxidation of 1-N -etheno-2’-deoxyguanosine by O
2
Lee, P.C.C., Rodgers, M.A.J. (1987). Laser flash photokinetic studies of Rose Bengal
sensitized photodynamic interactions of nucleotides and DNA. Photochem. Photobiol.
4
5, 79–86.
Lissi, E.A., Encinas, M.V., Lemp, E., Rubio, M.A. (1993). Singlet Oxygen O
bimolecular process. Solvent and compartmentalization effects. Chem. Rev. 93,
99-723.
Martinez, G.R., Ravanat, J-L., Medeiros, M.H.G., Cadet, J., Di Mascio, P. (2000). Synthesis
1
2
g
( Δ )
6
1
8
of a naphthalene endoperoxide as a source of O-labeled singlet oxygen for mechanistic
studies. J. Am. Chem. Soc. 379, 10212–10213.
1
8
Martinez, G.R., Medeiros, M.H.G., Ravanat, J-L., Cadet, J., Di Mascio, P. (2002). [ O]-
labeled singlet oxygen as a tool for mechanistic studies of 8-oxo-7,8-dihydroguanine
oxidative damage: Detection of spiroiminodihydantoin, imidazolone and oxazolone
derivatives. Biol. Chem. 383, 607-617.
Martinez, G.R, Ravanat, J-L., Cadet, J., Medeiros, M.H.G., Di Mascio, P. (2007).
Spiroiminodihydantoin nucleoside formation from 2 -deoxyguanosine oxidation by
1
8
[
O-labeled] singlet molecular oxygen in aqueous solution. J Mass Spectrom. 42,
326–1332.
1
Medeiros, M.H.G. (2009). Exocyclic DNA adducts as biomarkers of lipid oxidation and
predictors of disease. Challenges in developing sensitive and specific methods for
clinical studies. Chem. Res. Toxicol. 22, 419-425.
Miyamoto, S., Martinez, G.R., Medeiros, M.H.G., Di Mascio, P. (2003a). Direct evidence of
1
singlet molecular oxygen [O ( Δ )] production in the reaction of linoleic acid
2
g
hydroperoxide with peroxynitrite. J. Am. Chem. Soc. 125, 4510-4517.
Miyamoto, S., Martinez, G.R., Medeiros, M.H.G., Di Mascio, P. (2003b). Singlet molecular
oxygen generated from lipid hydroperoxides by the Russell mechanism: studies using
1
8
O-labeled linoleic acid hydroperoxide and monomol light emission measurements, J.
Am. Chem. Soc. 125, 6172-6179.
Miyamoto, S., Martinez, G.R., Medeiros, M.H.G., Di Mascio, P. (2014). Singlet molecular
oxygen generated by biological hydroperoxides. J. Photochem. Photobiol. B Biol. 139,
2
4-33.
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2006). Linoleic acid hydroperoxide reacts with hypochlorous acid, generating peroxyl
radical intermediates and singlet molecular oxygen. Proc. Nat. Acad. Sci. USA 103,
93-298.
(
2
Ravanat, J-L., Martinez, G.R., Medeiros, M.H.G., Di Mascio, P., Cadet, J. (2006). Singlet
oxygen oxidation of 2′-deoxyguanosine. Formation and mechanistic insights.
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(
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2001). Damage to isolated DNA mediated by singlet oxygen. Helv. Chim. Acta 84,
702–3709.
2
2
Sattsangi, P.D., Leonard, N.J., Frihart, C.R. (1977). 1,N -Ethenoguanine and N ,3-
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1
Oxidation of 1-N -etheno-2’-deoxyguanosine by O
2
Speina, E., Cieśla, J.M., Wójcik, J., Bajek, M., Kuśmierek, J.T., Tudek, B. (2001). The
6
pyrimidine ring-opened derivative of 1,N -ethenoadenine is excised from DNA by the
Escherichia coli Fpg and Nth proteins. J. Biol. Chem. 276, 21821–21827.
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6
1
,N -ethenoadenine. Mutat. Res. 531, 2015-217.
Tudek, B., Zdżalik-Bieleckac, D., Tudek, A., Kosickib, K., Fabisiewicze, A., Speina, E.
2017). Lipid peroxidation in face of DNA damage, DNA repair and other cellular
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processes. Free Radic. Biol. Med. 107, 77-89.
Wilkinson, F. (1993). Quantum yields for the photosensitized formation of the lowest
electronically excited singlet state of molecular oxygen in solution. J Phys Chem
Reference Data 22, 113.
Yip, K.F., Tsou, K.C. (1973). Synthesis of fluorescent adenosine derivatives. Tetrahedron
Lett. 33, 3067-3090.
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Oxidation of 1-N -etheno-2’-deoxyguanosine by O
2
Tables and figures
1
Table 1
Quenching constants of O
2
(k
t
= k
r
+ k
q
).
6
-1 -1
Compound
k
t
(10 M s )
5.39
dGuo
8
-oxodGuo
58.7
2
1
1
,N -εdGuo
2.35
6
,N -εdAdo
4.38
4
3
,N -εCyt
2.66
NaN
3
528
The values were obtained by Stern-Volmer plots.
Figure 1
Chemical structure of some etheno-adducts of nucleosides, dR=2-deoxyribose.
6
4
2
(
a) 1,N -etheno-2’-deoxyadenosine; (b) 3,N -etheno-2’-deoxycytidine; (c) N ,3-etheno-2’-
2
deoxyguanosine; (d) 1,N -etheno-2’-deoxyguanosine.
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Oxidation of 1-N -etheno-2’-deoxyguanosine by O
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Oxidation of 1-N -etheno-2’-deoxyguanosine by O
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Figure 2
(a) HPLC-ESI-MS/MS Analysis of the crude mixture after incubation of 1,N -
2
εdGuo with DHPNO
2
. Chromatograms obtained by HPLC analysis of the incubation of 1,N -
1
εdGuo with O
2
generated by DHPNO
2
. Monitoring at 260 nm in a photodiode array detector
and selecting the ion m/z 326, 268, 296 and 292 in full scan mode in the mass spectrometer
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Oxidation of 1-N -etheno-2’-deoxyguanosine by O
2
(
MIS – monitoring of the ion selected). (b) Mass spectra of the products A, B and C,
respectively. (c) UV spectra obtained by detection in photodiode array spectrophotometer of
2
HPLC for the generated products A, B and C after incubation of 1,N -εdGuo with DHPNO
2
.
(
d) UV spectra obtained by detection in photodiode array spectrophotometer of HPLC for the
2
2
product B generated after incubation of 1,N -εdGuo with DHPNO and an authentic sample of
dGuo.
2
Figure 3
HPLC-PDA analysis of 1,N -εdGuo photooxidation.
2
Compared was the photooxidation of 1,N -εdGuo in the presence of Rose Bengal (pink trace)
and the same reaction after incubation for one week at room temperature in the dark (black
trace) using gradient mode separation in reverse-phase [ultrapure water (A) and acetonitrile
(
B): 0 – 5 min, 5% B; 20 min, 10% B; 25 min, 5% B; 30 min, 5% B].
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Oxidation of 1-N -etheno-2’-deoxyguanosine by O
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Figure 4
Multiple-stage mass spectrometry (MSn) of product A.
The compound A was isolated by HPLC and after lyophilization, it was analyzed by mass
spectrometry with electrospray ionization at atmospheric pressure ionization using a multiple
stage mass spectrometer.
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Oxidation of 1-N -etheno-2’-deoxyguanosine by O
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Figure 5
(A) 1D selective TOCSY (A and B) and full H-NMR (C) spectra. The key
2
signals from the etheno diol hydrogens (a) and (b) formed along photooxidation of 1-N -
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Oxidation of 1-N -etheno-2’-deoxyguanosine by O
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εdGuo in the presence of Rose Bengal, corresponding to compound A. Proton signal selected
to magnetic excitation (a) and closest correlated signal (b) 1D TOCSY spectra were collected
at 100 ms of mixing time. (B) Edited HSQC NMR spectrum; the positive phase (black)
1
13
2
correspond to CH, and the negative phase (red) correspond to CH . H/ C NMR signals of
2
the crude mixture after photooxidation of 1-N -εdGuo in the presence of Rose Bengal.
2
Figure 6
εdGuo with DHPN O
Monitored was the absorbance at 260 nm in a photodiode array detector, selecting the ion m/z
98 (MIS) in full scan mode, the neutral loss of 116, and the ion m/z 182 (MIS) from the
daughters of 298 in the mass spectrometer.
HPLC-ESI-MS/MS analysis of the crude mixture after incubation of 1,N -
1
8
2
.
2
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Oxidation of 1-N -etheno-2’-deoxyguanosine by O
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1
Scheme 1
A possible mechanism of the reaction between 1,N -εdGuo and O
2
.
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Oxidation of 1-N -etheno-2’-deoxyguanosine by O
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Supplementary table and figures
Figure S1
(A) HPLC elution profile on a C-18 column of the crude mixture after
incubation of dGuo with chloroacetaldehyde at different pH. (B) UV spectra obtained for the
product formed at pH 6.5 and 8.0 and with retention time of 13.8 min on HPLC separation.
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Oxidation of 1-N -etheno-2’-deoxyguanosine by O
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Figure S2
Chromatograms obtained by HPLC analysis of the control sample and after
2
1
incubating 1, N -εdGuo with O
2
generated by photosensitization with MB.
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Oxidation of 1-N -etheno-2’-deoxyguanosine by O
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Figure S3
incubation of 1,N -εdGuo with O
60 nm and by mass spectrometry selecting the ion m/z 326, 268, 296 and 292 (MIS) in full
Chromatograms obtained by HPLC analysis of the products resultant from the
2
1
2
generated by photosensitization with MB. Monitoring at
2
2
scan mode. Mass spectra of the products A, B, C and 1,N -εdGuo.
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Oxidation of 1-N -etheno-2’-deoxyguanosine by O
2
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Figure S4
HR-MS of RB-photosensitization of 1,N -εdGuo. HPLC-ESI-MS/MS analysis
2
of photooxidation of 1-N -εdGuo in the presence of Rose Bengal using MIS mode selecting
2
the parent ions m/z 326 (A), m/z 268 (B), m/z 296 (C), and m/z 292 (1- N -εdGuo) with
fragments in m/z 210, m/z 152, m/z 180 and m/z 176, respectively. It was used gradient mode
separation condition in reverse-phase (ultrapure water (A) and ACN (B) : 0 – 5 min, 5% B; 20
min, 10% B; 25 min, 5% B; 30 min, 5% B.
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