A single nuclease-resistant linkage in DNA as a versatile tool for the characterization of DNA lesions: Application to the guanine oxidative lesion g+34 generated by metalloporphyrin/KHSO5 reagent

Article abstract of DOI:10.1021/tx300319y

The oxidation of an oligonucleotide containing a single nuclease-resistant phosphodiester link, a stereoisomerically pure methylphosphonate, by manganese (Mn-TMPyP) or iron (Fe-TMPyP) porphyrin associated to KHSO₅ allowed the isolation and characterization of a guanine lesion corresponding to an increase of mass of 34 amu as compared to guanine ( G+34 ), namely, 5-carboxamido-5-formamido-2-iminohydantoin. Enzymatic digestion of the damaged oligonucleotide afforded, apart from the undamaged nucleotide monomer pool, a unique dinucleotide doubly modified with a methylphosphonate and an oxidized guanine base that is suitable for NMR analysis. The method can be applied to the study of any DNA lesion. More importantly, the method can be extended to the analysis of DNA damage in a sequence context. Any preselected residue in a DNA sequence may be individually analyzed by the easy introduction of a single nuclease-resistant link at the 3′- or 5′-position.

Full text of DOI:10.1021/tx300319y

Article
pubs.acs.org/crt
A Single Nuclease-Resistant Linkage in DNA as a Versatile Tool for
the Characterization of DNA Lesions: Application to the Guanine
Oxidative Lesion “G+34” Generated by Metalloporphyrin/KHSO5
Reagent
Agnieszka Tomaszewska, Sophie Mourgues, Piotr Guga, Barbara Nawrot, and Genevieve Pratviel*
̀
S
* Supporting Information
ABSTRACT: The oxidation of an oligonucleotide containing
a single nuclease-resistant phosphodiester link, a stereoisomeri-
cally pure methylphosphonate, by manganese (Mn-TMPyP)
or iron (Fe-TMPyP) porphyrin associated to KHSO5 allowed
the isolation and characterization of a guanine lesion
corresponding to an increase of mass of 34 amu as compared
to guanine (“G+34”), namely, 5-carboxamido-5-formamido-2-
iminohydantoin. Enzymatic digestion of the damaged
oligonucleotide afforded, apart from the undamaged nucleo-
tide monomer pool, a unique dinucleotide doubly modified
with a methylphosphonate and an oxidized guanine base that is
suitable for NMR analysis. The method can be applied to the study of any DNA lesion. More importantly, the method can be
extended to the analysis of DNA damage in a sequence context. Any preselected residue in a DNA sequence may be individually
analyzed by the easy introduction of a single nuclease-resistant link at the 3′- or 5′-position.
versatile method to isolate dinucleotide units (instead of
nucleosides) by enzymatic digestion of a damaged double-
stranded oligonucleotide. It is based on the preparation of a
self-complementary oligonucleotide carrying a single nuclease-
resistant phosphodiester linkage at a precise site on the
sequence. The nuclease-resistant link must be located at the 3′-
or 5′-position of the nucleoside unit in the DNA sequence that
is considered as the target of the DNA-damaging reaction. After
DNA damage and enzymatic hydrolysis, the nuclease-resistant
dinucleotide containing the damaged residue (and the vicinal
nondamaged residue) can be isolated and characterized by
NMR.
We applied this method to the structural characterization of a
guanine oxidation product formed by the reaction of a DNA
oxidizing reagent based on a metalloporphyrin/KHSO5 system
with an oligonucleotide modified with a single stereoisomeri-
cally pure methylphosphonate linkage. We found that the
product corresponds to the previously identified 5-carbox-
amido-5-formamido-2-iminohydantoin (2-Ih) formed with
other oxidizing reagents.9,10 Additionally, labeling studies clarify
the mechanism of its formation and support epoxidation as the
oxidation event leading to this particular guanine lesion under
our experimental conditions.
INTRODUCTION
■
Oxidative damage of DNA can be induced by ionizing
radiation, photosensitization, or various chemical oxidants
giving rise to a number of DNA lesions such as oxidized
nucleic acid bases, oxidized sugar units, and DNA breaks.1−3
DNA damage may also be caused by various electrophiles
(alkylating agents and metal complexes) that lead to chemically
modified bases.3,4 DNA damage plays a key role in mutagenesis,
carcinogenesis, and cellular aging and is also considered as the
main mechanism of action of anticancer drugs. Consequently, it
is important to identify at a molecular level the DNA lesions
that may be responsible for biological effects. We report a
strategy that will facilitate the analysis of modified DNA and the
study of DNA repair.
The modifications of DNA bases can be characterized by
nuclear magnetic resonance (NMR) of nucleosides either after
hydrolysis of damaged high molecular weight DNA or by
performing in vitro damage on individual nucleosides.
However, some DNA lesions may escape detection at the
nucleoside level because of low UV absorbance or inappro-
priate chromatographic properties. The characterization of such
lesions may be easier on dinucleotides5−7 or trinucleotides.8
The UV absorbance of unmodified residues of di(tri)-
nucleotides facilitates the detection on HPLC chromatograms,
and different chromatographic properties may allow a better
separation. On the other hand, di(tri)nucleotide models are still
not considered as ideal substrates for the study of DNA
damage. A double-stranded DNA oligonucleotide model is
physiologically more relevant. Because structural analysis of
damaged oligonucleotides by NMR is not trivial, we designed a
EXPERIMENTAL PROCEDURES
Chemicals. H2O was of Milli-Q grade (Millipore). Oligodeoxy-
ribonucleotide (ODN) 5′-d(CAGCTG) was from Eurogentec SA
■
Received: July 13, 2012
Published: October 1, 2012
© 2012 American Chemical Society
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(Belgium) and purified by HPLC if necessary. The concentration was
determined by UV at 260 nm, ε = 55000 M−1 cm−1. Manganese
porphyrin was prepared as described.11 Nuclease P1 from Penicillium
citrinum was purchased from Sigma-Aldrich. KHSO5 (triple salt
2KHSO5·KHSO4·K2SO4) was from Aldrich. The 18O-labeled water
(97.2 atom %) was from Eurisotop, France.
concentrated by lyophilization and subjected to a second chromatog-
raphy on a Hichrom Inert (C18 ODS2, 5 μm, 250 mm × 10 mm)
eluted in the gradient mode with 50 mM TEAA buffer, pH 6.5, and
acetonitrile: 6−10% acetonitrile in 40 min followed by 10−15%
acetonitrile in 5 min. The flow rate was 4.7 mL/min, and detection was
at λ = 260 nm. The collected fractions (separated oxidized strands)
were desalted one by one on SepPak cartridges (elution with H2O/
acetonitrile) and lyophilized. The oxidized oligonucleotide 5′-
d[CAGCTPMe(G+34)] eluted at Rt = 4 and 4.5 min for the fast-
and slow-eluting isomers, respectively. The fast-eluting, minor isomer
fraction contained 7.5 OD (∼150 nmol), and the slow-eluting, major
isomer contained 18 OD (∼360 nmol). The purity of the products was
checked by analytical LC/ESI-MS.
Enzymatic Digestion. Typically, the purified oligonucleotide
carrying the damaged residue at G6, 5′-d[CAGCTPMe(G+34)] (3 OD,
∼6 nmol), was incubated with 1 unit of nuclease P1 in 30 mM sodium
acetate buffer, pH 5.4, for 5 min at 37 °C. The total reaction volume
was 100 μL. The enzyme was extracted by dichloromethane, and the
aqueous phase was injected on HPLC column for the isolation of the
modified dinucleotide 5′-d[pTPMe(G+34)] by chromatography. The
collected fractions from several hydrolysis reactions were pooled and
lyophilized. The purified dinucleotide of interest 5′-d[pTPMe(G+34)]
was stored dry at −20 °C before NMR analysis.
Synthesis of P-Stereopure Methylphosphonate Oligonu-
cleotide. Stereochemically pure methylphosphonate-modified oligo-
nucleotide, 5′-d(CAGCTPMeG) (“isomer I”, 400 OD, about 7 μmol
and 12 mg), was prepared with an OligoPilot II synthesizer
(Pharmacia Biotech, Sweden) using commercially available phosphor-
amidite monomers (Glen Research, Sterling, VA) and synthesis
protocols provided by the manufacturer. Nine consecutive syntheses at
the 40 μmol scale each furnished ca. 6000 OD units of crude product.
One has to notice that the coupling of the methylphosphonate
monomer was significantly less effective despite an extended time of
condensation. Because of intrinsic instability of methylphosphonate
linkages under alkaline conditions, the deprotection of nucleobases
was performed in concentrated ammonia at 55 °C for 4 h only. The
crude product was fractionated by means of preparative RP HPLC (a
column Pursuit XR, C18, 10 μm, 250 mm × 21.4 mm) with a gradient
of acetonitrile in TEAB (3%/min, rt ca. 10 min), and ca. 1000 OD
units of desired oligonucleotide was collected as a mixture of two P-
diastereoisomers (RP and SP). The mixture was subjected to
preparative anion exchange chromatography (a column BioBasicAX,
5 μm, 150 mm × 21.2 mm) with a nonlinear gradient of 1 M KCl in
20 mM NaH2PO4 (0−50% over 10 min, then 50−100% over 4 min,
then isocratic at 100% for 10 min, rt 14 and 15 min), which yielded
almost pure P-diastereomers. They were further desalted using the
aforementioned preparative C18 column eluted with a gradient of
acetonitrile in TEAB (1%/min).
NMR. NMR spectra were collected using a Bruker Avance 600
spectrometer equipped with a 5 mm triple resonance inverse Z-
gradient cryoprobe. All chemicals shifts for 1H are relative to the
residual HOD resonance (relative to tetramethylsilane). 1H NMR
spectra were collected at 298 K in D2O. Presaturation was used to
1
suppress the residual water signal. All of the H and 13C signals were
assigned on the basis of chemical shifts, spin−spin coupling constants,
splitting patterns, and signal intensities and by using 1H−1H total
1
correlation spectroscopy (TOCSY), H−1H transverse rotating-frame
HPLC and HPLC Coupled to Electrospray Mass Analysis (LC/
ESI-MS). The HPLC apparatus was an Agilent 1200 system. The
oligonucleotide reactions were analyzed on a reverse-phase Uptishere
column (C18, 5 μm, 250 mm × 4.6 mm from Interchrom, France)
eluted in the gradient mode: linear gradient from 4 to 15% of
acetonitrile in 10 mM triethylammonium acetate buffer (TEAA), pH
6.5, for 60 min. The flow rate was 0.5 mL min−1, and detection was at
λ = 260 nm. LC/ESI-MS analyses were carried out under the same
chromatographic conditions with an Agilent 1640 or a Qtrap AB Sciex
spectrometer (electrospray ionization in the negative mode).
1
Overhauser enhancement spectroscopy (T-ROESY), H−13C hetero-
nuclear multiple-quantum correlation (HMQC), and 1H−13C
heteronuclear multiple bond correlation (HMBC) experiments. The
2D T-ROESY spectra were acquired with a mixing time of 300 ms, and
TOCSY spectra were recorded with a spin-lock time of 60 ms.
Typically, 2048 t2 data points were collected for 256 t1 increments.
Spectra processing was performed using Bruker Topspin software.
RESULTS AND DISCUSSION
■
Analytical Oxidation of 5′-d(CAGCTG) and 5′-d(CAGCTPMeG).
Self-complementary oligonucleotide 5′-d(CAGCTG) or 5′-d-
(CAGCTPMeG) (20 μM strand concentration, 10 μM duplex
concentration) was allowed to anneal in 50 mM phosphate buffer,
pH 8, and 100 mM NaCl at 0 °C for 15 min and was then reacted with
Mn-TMPyP (or Fe-TMPyP) (10 μM) and KHSO5 (100 μM). The
concentrations are final concentrations in a reaction volume of 100 μL.
The reaction lasted for 10 min at 0 °C. The addition of excess HEPES
buffer with respect to KHSO5 stopped the reaction. When the reaction
was carried out in labeled water in the case of 5′-d(CAGCTG), the
reaction medium was lyophilized before the addition of the minimum
volume of a solution of KHSO5 prepared in nonlabeled water. The
final content of 18O-labeled water in the reaction medium was 96.7
atom %. LC/ESI-MS analyses were carried out on a small range of m/z
values for better resolution of isotopic patterns (905−920 amu).
Oxidation of 5′-d(CAGCTPMeG) on a Preparative Scale for
Isolation of 5′-d[CAGCTPMe(G+34)]. Stereochemically pure 5′-
d(CAGCTPMeG) (isomer I, 50 OD, ∼10 μmol, 20 μM strand
concentration) was reacted with Fe-TMPyP (20 μM) and KHSO5
(200 μM) in 50 mM phosphate buffer, pH 8, and 100 mM NaCl at 0
°C. The concentrations are final concentrations. The reaction lasted 20
min and was performed in a final volume of 50 mL. Individual
reactions were pooled together and desalted on a SepPak C18 (5 g
cartridge from Waters), and the concentrated material eluted with
acetonitrile/H2O (30:70, v/v) was lyophilized.
DNA Oxidative Damage by Manganese Porphyrin
Associated to KHSO5. The manganese porphyrin meso-
tetrakis(4-N-methylpyridiniumyl)porphyrinatomanganese(III)
(Mn-TMPyP)/KHSO5 is a powerful oxidation reagent
(Scheme 1).12 It has been used as a tool to identify DNA
lesions under oxidative stress.5−7,13−18 The mechanism of
action of this oxidation catalyst is reminiscent of cytochrome
P450 enzymes since it involves as an active species a high-valent
metal-oxo entity in the form of a MnVO entity (Scheme 2).
On DNA, the metal-oxo porphyrin is able to perform oxidation
Scheme 1. Structure of the Metalloporphyrins Mn-TMPyP
and Fe-TMPyP
Isolation of 5′-d[CAGCTPMe(G+34)]. A first chromatographic
separation was carried out on a C18eq 400 mg cartridge from
Macherey-Nagel eluted with 50 mM TEAA buffer, pH 6.5, and
acetonitrile (90:10, v/v). The collected fraction (oxidized strands) was
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by NMR on the nucleobase9 or nucleos(t)ides.10 It was
obtained with epoxidizing agents such as dimethyldioxirane9
and m-chloroperbenzoic acid.10 Resolution of diastereomers in
the oxidized nucleos(t)ides and 5-mer oligonucleotide indicates
that the oxidized base contains a chiral carbon.10 The
compound may be formed by two different mechanisms
depending on the oxidizing reagent used: epoxidation of the
C4−C5 bond of guanine9,10 or guanine oxidation by electron
transfer.23 In both cases, it arises from a rearrangement of an
initial intermediate, 1 in the case of electron transfer and 3 in
the case of epoxidizing reagents (Scheme 3). The rearrange-
ment is reminiscent of the acyl shift previously reported in the
mechanism of formation of another oxidative guanine lesion,
spiroiminodihydantoin (Sp),24−28 which corresponds to a four-
electron oxidation product of guanine instead of a two-electron
oxidation product in the case of G+34. Alternatively, compound
2 was also recently described to form through the coordination
of a nickel and a copper complex at N7 of guanine in the
presence of KHSO5 and H2O2, respectively.21,22
We decided to isolate the lesion of interest from the
oxidation of small DNA duplex using a modified oligonucleo-
tide with a single nuclease-resistant internucleotide link 5′ to
the guanine residue targeted for damage. In the case of short
oligonucleotide duplexes subjected to oxidation in vitro, the
most accessible residues are located at external base pairs. The
natural self-complementary oligonucleotide 5′-d(CAGCTG)
affords an external CG base pair, which was the site of major
oxidation by Mn-TMPyP/KHSO5. From liquid chromatog-
raphy coupled to negative electrospray mass spectrometry
analysis (LC/ESI-MS) of the oxidation reaction, the oxidized
strands contained either an oxidized guanine residue (guanine
lesions) or an oxidized sugar (ribonolactone) (Figure S1 and
Table S1 in the Supporting Information). The control
experiment in the presence of KHSO5 alone does not show
any degradation. Oligonucleotide 5′-d(CAGCTG) contains
two guanines (G3 and G6). The determination of the site of
oxidation is possible by piperidine treatment of oxidized DNA.
Most oxidized guanine residues are alkali-labile sites, and a
DNA break is generated at the site of the oxidized guanine.
Clearly, the 5-mer fragment 5′-d(CAGCT) sequence with a 3′-
phosphate group, 5′-d(CAGCTp), was the major species from
piperidine treatment of oxidized 5′-d(CAGCTG) after Mn-
TMPyP/KHSO5 oxidation (not shown). Consequently, we
decided to position a nuclease-resistant link at the junction
between T5 and terminal guanine residues to isolate oxidative
damage products at G6.
Scheme 2. Formation of an Active Metal-Oxo Porphyrin by
Reaction of KHSO5 with Metalloporphyrins Mn-TMPyP or
Fe-TMPyP (M = Mn or Fe)
a
a
The metal-oxo entity (MVO) is a strong oxidant and is capable of
oxygen atom transfer and/or electron transfer reactions like the active
intermediate of heme enzymes. The porphyrin macrocycle is
simplified. S refers to a substrate.
by electron transfer from guanine bases as well as oxygen atom
transfer at 2-deoxyribose units.16,18,19 Previously, among the
products of guanine oxidation, we observed a product with a
molecular mass corresponding to an increase of 34 amu as
compared to the mass of the nondamaged DNA.14,16 This
compound, referred to as “G+34”, was only identified by mass
spectrometry, and a structure was proposed (1 in Scheme 3) on
Scheme 3. Two Possible Mechanisms of Guanine Oxidation
a
Leading to the G+34 Lesion
Oxidation of the Natural 5′-d(CAGCTG) Oligonucleo-
tide by Iron Porphyrin Associated to KHSO5. The iron
porphyrin meso-tetrakis(4-N-methylpyridiniumyl)-
porphyrinatoiron(III) (Fe-TMPyP)/KHSO5 is also a powerful
oxidation reagent with similar oxidation properties as compared
to its manganese analogue.12 However, the two catalytic
systems do not show exactly the same reactivity.12 In the
present work, oxidation of 5′-d(CAGCTG) with Fe-TMPyP/
KHSO5 system afforded an oxidation profile including a higher
proportion of the so-called G+34 lesion (Figure S2 and Table
S2 in the Supporting Information). The G+34 lesions obtained
with the two different porphyrin catalysts were identical
according to their retention times on HPLC trace (coinjection
not shown). Therefore, in the present work, we decided to use
the Fe-TMPyP/KHSO5 system instead of the Mn-TMPyP/
KHSO5 system for the isolation of G+34.
a
C5 and C8 of G are indicated. dR stands for 2′-deoxyribose unit. ET,
electron transfer.
the basis of labeling studies implying guanine oxidation by
electron transfer followed by the attack of a water molecule at
carbon C5 of a guanine cationic intermediate. The aim of the
present study was to further characterize this guanine lesion.
The G+34 lesion has also been observed by others, using
different DNA-oxidizing agents, copper complexes,20,21 nickel
complexes,22 epoxidizing agents,9,10 and one-electron oxi-
dants.23 The 2-Ih structure (2 in Scheme 3) was determined
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Figure 1. (A) HPLC trace of the oxidation of 5′d(CAGCTPMeG) by Fe-TMPyP/KHSO5 (C18 reverse phase). The single-stranded 6-mer elutes at
48 min. Oxidized oligonucleotides elute at different retention times. They carry different lesions, namely, G+34, spiroiminohydantoin (Sp), abasic
site (ab), imidazolone (Iz), and oxidized guanidinohydantoin (DGh). (B) Structure of the observed DNA lesions resulting from guanine oxidation.
“M+34” refers to a nonidentified lesion corresponding to an increase of 34 amu with respect to the nondamaged oligonucleotide.
Stereoisomerically Pure Single Methylphosphonate-
Modified Oligonucleotides. In the context of seeking
guanine oxidative lesions, care must be taken in the choice of
the nuclease-resistant phosphodiester modification introduced
in DNA. Phosphorothioates are transformed to phosphates
under oxidative conditions29 and were discarded. Methyl-
phosphonate linkage was selected because it proved resistant
under oxidation conditions, and single-methylphosphonate
modified oligonucleotides can often be separated into P-
diastereomerically pure forms. The synthesis of 5′-d-
(CAGCTG) oligonucleotide with a methylphosphonate link
between T5 and G6, 5′-d(CAGCTPMeG) was performed using
the phosphoramidite approach, followed by chromatographic
separation of P-diastereomers on an anion-exchange column.
That procedure furnished fast-eluting (400 OD) and slow-
eluting (338 OD) P-diastereomeric compounds, further
referred to as the isomers I and II, respectively. The
stereochemistry of the methylphosphonate link was not
determined. According to Tm values of 22 and 29 °C for
isomer I duplex and isomer II duplex, respectively, one may
propose that isomer I is the RP diastereomer and isomer II is
the SP diastereomer of methylphosphonate.30
Both duplexes composed of two strands of either isomer I or
isomer II allowed us to observe the typical oxidation products
of guanine at the external base pair position. Isomer I was used
in the present study. It was analyzed by LC/ESI-MS. The
doubly charged ion observed at m/z = 894.0 amu was
consistent with the calculated m/z = 894.1 amu (Figure S3 in
the Supporting Information).
Oxidation of Methylphosphonate-Modified 5′-d-
(CAGCTPMeG) with Fe-TMPyP/KHSO5 and Mn-TMPyP/
KHSO5 Systems. The duplex oligonucleotide composed of
two strands of the self-complementary 5′-d(CAGCTPMeG) (10
μM duplex DNA) was incubated in 50 mM phosphate buffer,
pH 8, and NaCl 100 mM, with Fe-TMPyP (10 μM) and
KHSO5 (100 μM) at 0 °C for 10 min. The reaction was
stopped by the addition of excess HEPES buffer31 when 30% of
the oligonucleotide was damaged. HPLC/ESI-MS analysis of
the reaction showed several types of oxidized oligonucleotide
strands eluting between 38 and 46 min before the residual
nondamaged oligonucleotide at 48 min (Figure 1A). The
lesions were attributed to known modified guanine residues,
including spiroiminohydantoin (Sp), oxidized guanidinohydan-
toin (DGh), imidazolone (Iz), and abasic site (Figure 1B and
Table 1). Most of them have been previously characterized
Table 1. LC/ESI-MS Analysis of the Oxidation of 5′-
d(CAGCTPMeG) by Fe-TMPyP/KHSO5
m/z (z = 2)
m/z (z = 2)
Rt (min)
obsd
lesion
calcd
38.8
40
911.2
911.0
909.9
909.9
911.1
874.5
827.4
895.8
894.0
G+34
G+34
Sp
911.1
911.1
910.1
910.1
911.1
874.6
827.6
896.1
894.1
41.5
42.2
Sp
43−44
M+34
Iz
abasic site
G+4
44.4
48
5′-d(CAGCTPMeG) (isomer
I)
from DNA oxidation by the Mn-TMPyP/KHSO5 sys-
tem5−7,13,14 except the guanine lesion with a mass correspond-
ing to an increase of 34 amu with respect to the molecular mass
of G. It is referred to as the G+34 lesion. Among the oxidized
oligonucleotide strands, two peaks appeared with a mass
corresponding to G+34 (m/z = 911.1) at 38.8 and 40 min.
They were referred to as fast- and slow-eluting G+34 isomers.
The fast-eluting compound was in a lower amount as compared
to the slow-eluting one (about 1/3 ratio) (Figure 1A). Another
product with a m/z signal also corresponding to an increase of
34 amu was observed at 43−44 min, “M+34” in Figure 1A.
However, the peak contained several nonseparated products
and was not studied in the present work (Table 1). The two
oxidized oligonucleotide strands (fast- and slow-eluting G+34
isomers) at 38.8 and 40 min were collected separately by
preparative HPLC from a larger scale preparation.
For comparison, the reaction of Mn-TMPyP/KHSO5 system
with 5′-d(CAGCTPMeG) under the same experimental
conditions led to the same products but in different proportions
(Figure S4 and Table S3 in the Supporting Information). As
observed on natural oligonucleotides, DGh was the major
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product of guanine oxidation, while G+34 was a minor
compound. Besides, only the slow-eluting G+34 oligonucleo-
tide could be detected at 40 min.
compared to that originating from the slow-eluting oligonucleo-
tide on the reverse-phase column.
The dinucleotide 5′-d[pTPMe(G+34)] carrying the methyl-
phosphonate nuclease-resistant link and the oxidized guanine G
+34 lesion was isolated in the case of the slow-eluting major
Hydrolysis of the Oxidized Methylphophonate Oligo-
nucleotides, 5′-d[CAGCTPMe(G+34)]. First, the nondamaged
methylphosphonate oligonucleotide, 5′-d(CAGCTPMeG) (iso-
mer I), was reacted with nuclease P1 at 37 °C in 30 mM acetate
buffer, pH 5.3, for 5 min. The rapid and complete hydrolysis
led to the formation of nucleoside dC, nucleotides dGMP and
dAMP with a 5′-phosphate group, as well as the dinucleotide
carrying the methylphosphonate link with a 5′-phosphate
group, 5′-d(pTPMeG) (Rt = 34 min, Figure 2A). Hydrolysis of
1
isomer oligonucleotide (Figure 2B) and analyzed by H and
1
13C NMR (Tables 2 and 3). Importantly, a H,13C HMBC
Table 3. 13C Resonances (δ, ppm) Extracted from
1
1
Correlations in H/13C HSQC and H/13C HMBC
Experiments for 5′-d[pTPMe(G+34)] Dinucleotide in D2O
2-deoxyribose
thymine
G+34
C1′
C2′
C3′
C4′
C5′
CH3
C5
T
85.0
88.2
37.0
38.4
77.1
70.9
84.2
84.2
64.2
65.7
11.7
G+34
79.2
cross-peak was observed between a carbon resonance at 79.2
ppm (attributed to the quaternary carbon) and a proton
resonance at 8.76 ppm (attributed to the aldehyde group) on
the modified base. Furthermore, a nuclear Overhauser effect
(NOE) between the proton at 8.76 ppm from the modified
base and the H2′H2′′ protons of the 3′-sugar at 2.57−2.64
ppm confirms that the proton at 8.76 ppm belongs to the
modified nucleoside. The proposed structure of the lesion is
shown in Scheme 4. A minor form of this compound can be
Scheme 4. Structure of 5′-d[pTPMe(G+34)] Dinucleotide
Modified with a Nuclease-Resistant Methylphosphonate and
Carrying the G+34 Lesion, Namely, 2-Ih
Figure 2. HPLC trace (λ = 260 nm, C18 reverse phase) of the
nuclease P1 hydrolysis of 5′-d(CAGCTPMeG) (A) and slow-eluting
and fast-eluting G+34 lesion carrying oligonucleotide strands (B and
C), respectively. The hydrolysis is complete, and the full-length ODN
is absent from the chromatogram. The nucleoside dimer carrying the
methylphosphonate bridge is observed at 35 (A) and 20 min (B) and
coelutes with dAMP at 15 min (C). X = impurity.
detected by an exchange correlation between the H-signal at
8.76 ppm with a signal at 8.22 ppm (accounting for 7−8%). As
previously proposed, this minor form might correspond to a
rotational isomer of the formyl group.10
The fast- and slow-eluting oligonucleotides at retention times
of 38.8 and 40 min, respectively (Figure 1A), and/or
dinucleotides carrying a G+34 lesion (Figure 2B,C) correspond
probably to two diastereoisomers due to the presence of a new
chiral carbon at the modified guanine residue. The stereo-
chemistry of the new chiral center on the oxidized guanine
residue could not be determined. This G+34-modified base
each of the fast- and slow-eluting G+34 lesion carrying
oligonucleotides led to the corresponding nuclease-resistant
dinucleotides but carrying the oxidized guanine lesion, namely,
d[TPMe(G+34)] with a 5′-phosphate group, 5′-d[pTPMe(G
+34)] (Figure 2B,C). The elution of the modified dinucleotides
followed the same order as the full-length oligonucleotides
from which they originated. The dinucleotide obtained from
the fast-eluting modified oligonucleotide eluted faster as
1
Table 2. H NMR Data (δ, ppm) for 5′-p-d[pTPMe(G+34)] Dinucleotide in D2O
2-deoxyribose
thymine
G+34
CHO
H1′
6.36 (dd), JHH = 5.8 Hz, JHH = 8.3 Hz
H2′, H2″
2.56−2.45
H3′
H4′
H5′, H5″
CH3
1.90
H6
P-CH3
3
3
T
5.14
4.38
4.05
7.73
3
1.68 (d), JHP = 17.7 Hz
3
3
G+34
5.66 (dd), JHH = 3.2 Hz, JHH = 7.1 Hz
2.64−2.57
4.44
4.11
4.11−4.18
8.76
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corresponds to the previously characterized 2-Ih.9,10 As in the
present work, the two isomers did not form in equal amounts.10
Mechanism of Formation of G+34 Lesion. The
mechanism of formation of the G+34 lesion with Mn-
TMPyP/KHSO5 and Fe-TMPyP/KHSO5 as oxidizing agent
was investigated by labeling studies. According to Scheme 3,
two new O-atoms are incorporated in the oxidized guanine
residue.
It is possible to discriminate between mechanisms involving
electron transfer and oxygen atom transfer by the analysis of
the origin of the oxygen atoms incorporated in G+34 when the
reaction is carried out in labeled water. Electron transfer should
lead to the incorporation of two O-atoms from H2O. O-atom
transfer should lead to incorporation of the O-atom from the
high-valent metal-oxo species of the activated metal-oxo
porphyrin at the former C5 of guanine.
KHSO5 oxidant, as a signal at m/z = 913.4 amu in labeled water
for electron transfer mechanism (incorporation of two 18O-
atoms at former C5 and C8 carbons of guanine), while the
mass signal should be composed of two species of equal
intensity with m/z = 912.4 (one 18O-atom incorporated at
former C8) and 913.4 amu (for 2 18O-atoms incorporated at
former C5 and C8) in labeled water. On the other hand, for Fe-
TMPyP/KHSO5 oxidant, an electron transfer mechanism
should lead to the incorporation of two 18O-atoms in G+34
as for Mn-TMPyP/KHSO5, giving a mass signal at m/z = 913.4
amu in labeled water. An oxygen atom transfer mechanism
should lead to a single mass signal at m/z = 912.4 amu
(incorporation of one 16O-atom at former C5 and one 18O-
atom at former C8).
Oxidation of the natural 5′-d(CAGCTG) oligonucleotide
was carried out in normal and in labeled water with Mn-
TMPyP/KHSO5 and with Fe-TMPyP/KHSO5. LC/ESI-MS
analysis of the reaction with Mn-TMPyP showed that the G+34
carrying oligonucleotide with an m/z signal at 911.4 amu (z =
2) in H216O (Figure 3A) appeared as a mixture of two species
Manganese porphyrin undergoes oxo-hydroxo tautomerism
at the MnVO in water.19,32 Thus, O-atom transfer in labeled
water should lead to the incorporation of an O-atom at the
former C5 of guanine in the form of 16O/18O in a 50/50 ratio
(Scheme 5). In the case of iron porphyrin, the formation of μ-
Scheme 5. Oxygen Atom Transfer Mediated by Manganese-
Oxo Porphyrin in H218O and Oxo-Hydroxo Tautomerism at
a
the MnVO Species
Figure 3. In-line mass spectrum of oxidized oligonucleotide strands
carrying the G+34 lesion in the oxidation of 5′-d(CAGCTG) with Fe-
and/or Mn-TMPyP/KHSO5 in H216O (A), Mn-TMPyP/KHSO5 in
H218O (B), and Fe-TMPyP/KHSO5 in H218O (C). Doubly charged
ions are observed (z = 2). Corresponding HPLC traces are shown in
Figure S1 (Mn) and Figure S2 (Fe) in the Supporting Information.
a
The porphyrin macrocycle is simplified. Black and white O-atom
corresponds to 18O- and 16O-atom, respectively.
oxo species in pH 8-buffered solution precludes the oxo-
hydroxo tautomerism. Thus, O-atom transfer mediated by
FeVO species should lead to the incorporation of one 16O-
atom at the former C5 of guanine (Scheme 6). On the other
of equal abundance in H218O (Figure 3B). The lesion
incorporated one O-atom from water and one O-atom from
KHSO5 (m/z = 912.4 amu) or two O-atoms from labeled water
(m/z = 913.4 amu). For Fe-TMPyP, the G+34-carrying
oligonucleotide showed an in-line mass spectrum with a single
mass signal at m/z = 912.4 amu when the reaction was
performed in H218O (Figure 3C). These results are compatible
with a mechanism involving an epoxide intermediate (3 in
Scheme 3) for both porphyrins. It was previously reported that
the G+34 lesion did not undergo exchange of O-atoms with
solvent,10 and we also carried out control experiments
consisting of incubation of the labeled lesion under different
conditions for several hours (water, chromatography solvents),
and no change in labeling occurred (not shown).
Scheme 6. Oxygen Atom Transfer Mediated by Iron-Oxo
a
Porphyrin in H218O
a
The porphyrin macrocycle is simplified. Black and white O-atom
corresponds to 18O- and 16O-atom, respectively.
The last guanine base (G6 residue) of the short duplex
formed with 5′-d(CAGCTG) is perfectly accessible for the
high-valent metal-oxo form of the porphyrins. In the chemistry
of oxygen atom transfer by metal-oxo porphyrins, a close
contact between the metal-oxo and the double-bond that is the
site of epoxidation is required. The accessibility of the external
guanine base on the chosen oligonucleotide may explain the
epoxidation mechanism.
hand, the oxygen atom incorporated at the former C8 of
guanine should always originate from the attack of a water
molecule after modification of the guanine heterocycle by the
oxidation event, whatever the porphyrin used.
Thus, the nonlabeled mass signal for the G+34-modified
natural oligonucleotide at m/z = 911.4 amu (z = 2) observed in
nonlabeled water should appear, in the case of Mn-TMPyP/
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(6) Seguy, C., Pratviel, G., and Meunier, B. (2001) Characterization
of an oxaluric acid derivative as a guanine oxidation product. Chem.
Commun., 2116−2117.
CONCLUSION
■
The oxidation of an oligonucleotide containing a single
nuclease-resistant internucleotide link, a stereoisomerically
pure methylphosphonate, by Mn-TMPyP/kHSO5 and Fe-
TMPyP/KHSO5 allowed the isolation and characterization of a
guanine lesion corresponding to an increase of mass of 34 amu
as compared to guanine (G+34). The lesion was attributed to
2-Ih. Enzymatic digestion of the damaged oligonucleotide
afforded, apart from the nucleotide monomer pool, a unique
dinucleotide doubly modified with a methylphosphonate and
an oxidized guanine base that was suitable for NMR analysis.
The method can be applied to the study of any DNA lesion.
More importantly, the method can be extended to the analysis
of DNA damage in a sequence context. Any preselected residue
in a DNA sequence may be individually analyzed for damage at
a molecular level by the easy introduction of a single nuclease-
resistant link adjacent to the target nucleoside.
(7) Chworos, A., Seguy, C., Pratviel, G., and Meunier, B. (2002)
Characterization of the dehydro-guanidinohydantoin oxidation prod-
uct of guanine in a dinucleotide. Chem. Res. Toxicol. 15, 1643−1651.
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Ravanat, J. L. (2006) Characterization of lysine-guanine cross-links
upon one-electron oxidation of a guanine-containing oligonucleotide
in the presence of a trilysine peptide. J. Am. Chem. Soc. 128, 5703−
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(9) Ye, W., Sangaiah, R., Degen, D. E., Gold, A., Jayaraj, K., Koshlap,
K. M., Boysen, G., Williams, J., Tomer, K. B., and Ball, L. M. (2006) A
2-iminohydantoin from the oxidation of guanine. Chem. Res. Toxicol.
19, 506−510.
(10) Ye, W., Sangaiah, R., Degen, D. E., Gold, A., Jayaraj, K., Koshlap,
K. M., Boysen, G., Williams, J., Tomer, K. B., Mocanu, V., Dicheva, N.,
Parker, C. E., Schaaper, R. M., and Ball, L. M. (2009) Iminohydantoin
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(11) Romera, C., Sabater, L., Garofalo, A., Dixon, I. M., and Pratviel,
G. (2010) Interaction of cationic nickel and manganese porphyrins
with the minor groove of DNA. Inorg. Chem. 49, 8558−8567.
(12) Meunier, B., Robert, A., Pratviel, G., and Bernadou, J., Eds.
(2000) Metalloporphyrins in Catalytic Oxidation and Oxidative DNA
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(13) Vialas, C., Pratviel, G., Claparols, C., and Meunier, B. (1998)
Efficient oxidation of 2′-deoxyguanosine by Mn-TMPyP/KHSO5 to
imidazolone dIz without formation of 8-oxo-dG. J. Am. Chem. Soc. 120,
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(14) Vialas, C., Claparols, C., Pratviel, G., and Meunier, B. (2000)
Guanine oxidation in double-stranded DNA by Mn-TMPyP/KHSO5:
5,8-Dihydroxy-7,8-dihydroguanine residue as a key precursor of
imidazolone and parabanic acid derivatives. J. Am. Chem. Soc. 122,
2157−2167.
(15) Angeloff, A., Dubey, I., Pratviel, G., Bernadou, J., and Meunier,
B. (2001) Characterization of a 5′-aldehyde terminus resulting from
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ASSOCIATED CONTENT
■
S
* Supporting Information
HPLC traces, structures, and tables of LC/ESI-MS analysis.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: +33(0)561333146. Fax: +33(0)561553003. E-mail:
genevieve.pratviel@lcc-toulouse.fr.
Funding
This work was partly supported by a joint program between
CNRS, France, and the Polish Academy of Sciences (Polonium
20110NE) and by ANR 06SEST11.
Notes
The authors declare no competing financial interest.
(16) Mourgues, S., Kupan, A., Pratviel, G., and Meunier, B. (2005)
Use of short duplexes for the analysis of the sequence-dependent
cleavage of DNA by a chemical nuclease, a manganese porphyrin.
ChemBioChem 6, 2326−2335.
ACKNOWLEDGMENTS
■
We acknowledge Gauthier Foucras and Elsa Moreau for
technical help; Dr. Yannick Coppel, Laboratoire de Chimie de
Coordination, CNRS, for NMR analyses; and Dr. Chantal
Zedde, service commun de chromatographie, Institut de
Chimie, Toulouse, for the purification of the oligonucleotides.
Dr. Milena Sobczak provided excellent technical assistance in
synthesizing methylphosphonate oligonucleotides. We thank
Prof. Wojciech J. Stec for invaluable discussions and permanent
interest in the project.
(17) Kupan, A., Sauliere, A., Broussy, S., Seguy, C., Pratviel, G., and
Meunier, B. (2006) Guanine oxidation by electron transfer: one-
versus two-electron oxidation mechanism. ChemBioChem 7, 125−133.
(18) Pitie,
hydrogen bonds by metal complexes. Chem. Rev. 110, 1018−1059.
(19) Pitie, M., Bernadou, J., and Meunier, B. (1995) Oxidation at
́
M., and Pratviel, G. (2010) Activation of DNA carbon-
́
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guanine oxidation by a dinuclear copper(II) complex at single
stranded/double stranded DNA junctions. Inorg. Chem. 45, 7144−
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