The Two Main DNA Lesions 8-Oxo-7,8-dihydroguanine and 2,6-Diamino-5-formamido-4-hydroxypyrimidine Exhibit Strongly Different Pairing Properties

Article abstract of DOI:10.1002/anie.200351287

A stable analogue (cFapydGuo) of the major oxidative DNA lesion FapydGuo was prepared. The direct comparison of the pairing properties of the oxidative lesion β-8-oxodGuo and β-cFapydGuo (see formulae) revealed significant differences in base pairing, ind

Full text of DOI:10.1002/anie.200351287

Angewandte
Chemie
DNA Lesions
The Two Main DNA Lesions 8-Oxo-7,8-
dihydroguanine and 2,6-Diamino-5-formamido-4-
hydroxypyrimidine Exhibit Strongly Different
Pairing Properties**
Matthias Ober, Uwe Linne, Johannes Gierlich, and
Thomas Carell*
Dedicated to Professor R. W. Hoffmann
on the occasion of his 70th birthday
DNA damage generated by the reaction of DNAwith reactive
oxygen species is one of the major reasons for mutations and
carcinogenesis.[1–4] Additionally, oxidative DNA damage is
believed to play a major role in the pathogenesis of ageing.[5–9]
Reactive oxygen species (ROS) are generated as side
products during aerobic respiration, a process that involves
the stepwise reduction of molecular oxygen to water. One of
the most dangerous reaction products is the OH radical,
which is a potent electrophilic oxidant able to oxidize DNA.[4]
In DNA, the OH radical reacts preferentially with guanine.
Guanine is also the major target for general oxidative damage
because it has the lowest oxidation potential among the four
bases.
In the last decade, a large number of guanine-derived
genome lesions produced either by direct oxidation or
reaction with OH radicals were isolated and character-
ized.[10,11] 8-Oxo-7,8-dihydroguanine (8-oxodGuo) and 2,6-
diamino-5-formamido-4-hydroxypyrimidine
(FapydGuo,
Scheme 1) were found as two main degradation products.[12,13]
They are currently believed to be predominantly responsible
for the biological effect of oxidative and OH-radical-induced
DNA damage.[14]
To study repair and the mutagenic effect of both lesions it
is essential to prepare oligonucleotides that contain these
lesions in defined positions. To this end, lesion phosphorami-
dites are needed, which allow their incorporation into DNA
by using machine-assisted DNA synthesis.[15] The guanine
derivative 8-oxodGuo is a stable compound, which early on
allowed its chemical synthesis and incorporation into
DNA.[16,17] Consequently, a wealth of chemical and biochem-
ical data are now available on this important lesion.[18,19]
Investigation of the FapydGuo lesion, in contrast, is
strongly hindered owing to its instability.[20,21] We recently
reported the synthesis of the FapydGuo lesion in a protected
form and discovered that the compound is relatively stable
[*] Prof. Dr. T. Carell, Dipl.-Chem. M. Ober, Dr. U. Linne,
Dipl.-Chem. J. Gierlich
Department of Chemistry, Philipps-Universität Marburg
Hans-Meerwein Strasse, 35032 Marburg (Germany)
Fax: (+49)6421-282-2189
E-mail: carell@staff.uni-marburg.de
[**] The work was generouslysupported bythe Volkswagen Stiftung, the
Fonds der Chemischen Industrie, and the Deutsche Forschungs-
gemeinschaft. We thank the Boehringer Ingelheim Fonds for a
predoctoral fellowship to M.O.
Angew. Chem. Int. Ed. 2003, 42, 4947 –4951
DOI: 10.1002/anie.200351287
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4947

Communications
therefore the base-pairing properties of the lesion fully intact.
For the standard nucleotides, it was proven that the oxygen-
to-methylene chemical mutation has a negligible effect on
duplex stability and duplex structure.[29–31] To analyze possible
effects of the O-to-CH2 chemicalmutation on the syn/anti
conformationalequiibl rium, which is so criticalfor the
mutagenic potentialof the 8-oxodGuo, we performed molec-
ular modeling and density functional calculations.[32] In both
cases we obtained almost the same, very small differences
between the syn and anti conformations of only about 5 to
32 kJmolꢀ1, indicating that the heterocycle rotates in both
ꢀ
compounds freely around the C1’ N glycoside bond. The anti
conformation is energetically somewhat more favorable, in
agreement with NOESY experiments performed with com-
pound 6 in CDCl3. The NOESY showed stronger cross-peaks
between the C1’-NH and the endo protons at C2’, C3’, and
C5’, thus indicating a larger population of the anti conforma-
tion. Most important, however, is the result that the O-to-CH2
chemical mutation has, if at all, only a minor effect on the syn/
anti conformationalequiilbrium.
For the synthesis of the cFapydGuo analogue (Scheme 2),
we started with the enantiomerically pure cyclopentylamine
1, which was prepared in six steps by following the synthetic
route recently reported by Cullis and Dominguez.[33] Coupling
of this building block with the acetyl-protected 2-amino-6-
chloro-5-nitro-4-oxopyrimidine (2)[34–36] furnished the cyclo-
pentane derivative 3. Protection of the hydroxy groups with
TBDMSClyielded compound 4. The following steps included
reduction of the nitro group to the amine 5 and trans-
formation of the amine into the formamide 6.[22] Cleavage of
the TBDMS groups was performed with HClin ethylacetate.
Conversion of the carbocyclic analogue of the b-FapydGuo
lesion 7 into the phosphoramidite 9 was possible by using
standard procedures. The fully deprotected cFapydGuo lesion
10 was synthesized in two steps from 6. Deacetylation of
compound 6 was performed with a 0.05m solution of K2CO3 in
methanol. Desilylation required treatment of the product
with HCl in THF. The building block 9 was finally incorpo-
rated into oligonucleotides using slightly modified standard
DNA synthesis procedures, which will be described in due
course. The coupling of the cFapydGuo unit was found to be
above 98%. Cleavage of the fully assembled DNA from the
solid support and of all protecting groups present at the
various nucleotides was performed by using a saturated
solution of ammonia in water/ethanol (3:1) at 158C. Intensive
studies with the monomer 6 showed that at 158C, cleavage of
the acetyl protecting group is highly selective. All DNA
strands that contained cFapydGuo were finally purified by
reversed-phase HPLC.
Scheme 1. Depiction of the oxidative DNA lesion 8-oxodGuo base
paired with dC and with dA, together with the oxidative DNA lesion
FapydGuo.
under physiological conditions.[22] However, the compound
was found to anomerize rapidly under conditions generally
required for chemicalDNA synthesis. Greenberg and co-
workers published the synthesis of a dinucleotide building
block, which allowed the incorporation of the FapydGuo
lesion as an a/b-mixture into DNA.[23,24] Over the last two
years, this achievement has allowed initial information to be
gathered on how FapydGuo affects the stability of DNA
duplexes and how it is replicated by polymerases.[25]
Herein we report the synthesis of a stabilized bioisosteric
FapydGuo analogue,[26] which can be inserted in DNA as the
pure “b” anomer. The mutagenic potential of all DNA lesions
is strongly connected to their base-pairing properties, which
can be estimated by using melting-temperature studies. Such
melting-point data of the 8-oxodGuo lesion showed efficient
base pairing with dC and dA.[27] A crystalstructure for the 8-
oxodGuo in DNA opposite dA clarified that the lesion pairs
with dA if the N–glycoside bond adopts the unusual syn
conformation as shown in Scheme 1.[28]
The structuralsimialrity of 8-oxodGuo and FapydGuo,
=
manifested in the C8 O carbonylgroup present in both
lesions, led to the suggestion that both lesions might have very
similar base-pairing properties. This belief was recently
supported by melting-point data obtained with a/b-
FapydGuo-containing DNA.[24,27] In contrast, our experi-
ments show that FapydGuo has a pairing behavior very
different from that of 8-oxodGuo. This result suggests a
completely different mutagenic effect of FapydGuo.
To circumvent the anomerization problem, we prepared a
stabilized, strictly bioisosteric lesion analogue, which contains
a cyclopentane skeleton instead of the 2’-deoxyribose sugar
backbone.[29] Replacement of the 2’-deoxyribose oxygen atom
by a methylene group leaves the heteroatom structure and
Analysis of the DNA was carried out by analytical HPLC
and MALDI-TOF mass spectrometry. The obtained data
proved that the cFapydGuo-containing oligonucleotides were
prepared in excellent yields. The oligonucleotides had, after
preparative HPLC purification, a finalpurity of > 98%,
necessary for detailed melting-point studies. Final proof that
the cFapydGuo was correctly inserted into the DNA strand
was obtained by an HPLC/MS/MS experiment. To this end,
the cFapydGuo-containing DNA strand was fully digested in
an enzyme mixture of phosphodiesterase II, nuclease P1,
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Chemie
7, or the 8-oxodGuo lesion and all four canon-
ical bases as counterbases. All 12 double
strands gave CD spectra, which are typicalfor
B-helical DNA.[38] The differences in the CD
spectra were found to be very small, thus
confirming that the compounds FapydGuo and
8-oxodGuo have only a very small effect on the
overall duplex structure.
The melting temperatures measured for all
DNA double strands are summarized in
Table 1. As expected, the guanine-containing
strand shows the highest melting temperature
in the matching dG:dC situation. All mis-
matches strongly destabilize the duplex by at
least 148C. The two lesions 8-oxodGuo and
cFapydGuo destabilize the double strands,
regardless of the counterbase. However, in
agreement with previous reports, 8-oxodGuo
pairs relatively well with both dC and dA.[27]
Both base pairs provide double strands with
melting points that are only 68C lower than that
of the undisturbed dG:dC duplex. The 8-
oxodGuo lesion is therefore able to form the
expected anti8-oxodGuo:dC and syn8-oxodG-
uo:dA base pairs, also in the oligonucleotide
duplexes analyzed in this study. Our data
therefore support the base-pairing schemes
shown in Scheme 1.
In contrast to currently accepted theory, we
find that the melting behavior of the cFa-
pydGuo is drastically different to that of 8-
oxodGuo. First, the destabilization of the
duplexes in the presence of this lesion is much
more pronounced. This is surprisingly also the
case if dC functions as the counterbase. For the
cFapydGuo:dC base pair, the melting temper-
ature measured was similar to that associated
with a mismatch! The data show that cFa-
pydGuo double strands have the highest melt-
ing point with the counterbase dT.[39] In fact, the
Scheme 2. Synthesis of the cFapydGuo lesion analogue phosphoramidite 9 and depic-
tion of the DNA double strands prepared for melting-point studies. a) DIPEA, DMF,
708C, 1.5 h, 72%; b) TBDMSCl, imidazole, DMF, 208C, 3 h, 88%; c) Pd/C, H2, EtOH,
208C, 2.5 h; d) HCOOH, EDC, DIPEA, DMF, 208C, 36 h, 70% for two steps; e) HCl/
EtOAc (5%), 0!208C, 1.5 h, 95%; f) DMTCl, lutidine, DMF, 08C, 3 h, 49%; g) DIPEA,
iPr2NPOCH2CH2CN+Clꢀ, THF, 08C, 30 min, 86%; h) K2CO3 (0.05m)/MeOH, 208C, 2 h,
90%; i) HCl/THF (5%), 0!208C, 1.5 h, 95%. DIPEA=diisopropylethylamine,
DMF=N,N-dimethylformamide, TBDMS=tert-butyldimethylsilyl, EDC=1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride, DMT=4,4’-dimethoxytrityl.
phosphodiesterase I, and alkaline phosphatase.[37] The HPLC
chromatogram obtained after the digestion showed five
peaks, of which four could be assigned to the four nucleobases
A, T, G, and C by mass spectrometry. The fifth, small and
broad peak, which almost co-eluted with cytosine, was
assigned to the cFapydGuo. The peak showed the same
retention time as the co-injected compound 10. The molecular
weight corresponding to the new peak was found to be
identicalto that of compound 10, and finally also the
fragmentation patterns of 10 and of the new HPLC peak
were identical. Thus, the reported DNA synthesis procedure
and the availability of the building block 9 in large amounts
allowed the preparation of stable cFapydGuo-containing
oligonucleotides in quantities sufficient for detailed physico-
chemicaland structuralinvestigations.
cFapydGuo:dT base pair seems to be as stable as the 8-
oxodGuo:dC and 8-oxodGuo:dA base pairs.
In addition, all the double strands in which cFapydGuo
faces one of the two purine bases feature an unusualdoubel
sigmoidalmetling behavior. The curves obtained for the
double strands cFapydGuo:dA and cFapydGuo:dG (red
Table 1: List of the melting temperatures measured for the twelve DNA
duplexes shown in Scheme 1.[a]
5’-d(GCGATXTAGCG)-3’
3’-d(CGCTAYATCGC)-5’
Tm [8C]
X=dG
X=8-oxodGuo
X=cFapydGuo
Y=dA
Y=dC
Y=dG
Y=dT
33.9
51.8
37.9
35.8
46.0
45.6
31.4
36.7
16.1 (54.9)
36.9
16.1 (54.9)
45.0
To analyze the melting behavior and hence the pairing
properties of b-cFapydGuo, we prepared the DNA double
strands depicted in Scheme 2. The DNA duplexes contain
either the canonicalbase dG, the cFapydGuo lesion analogue
[a] Conditions: Tris /HCl, pH 7.4 (0.01m), NaCl (0.15m), DNA (3 mm).
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dramatically different pairing properties, which suggests that
their mutagenic properties vary as well.
Received: February 27, 2003
Revised: May 21, 2003 [Z51287]
Keywords: DNA damage · DNA · electron transfer · nucleotides ·
.
oxidative lesions
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curves), together with the corresponding melting curves of 8-
oxodGuo:dG and 8-oxodGuo:dA (blue curves) are depicted
in Figure 1. For cFapydGuo:dG and cFapydGuo:dA a first
very low melting point is observed at about 168C, which is not
observed in the experiments performed with the 8-oxodGuo
lesion. A second, much weaker transition is observed at
higher temperatures. This second transition cannot be
explained currently. One explanation could be that the
strongly destabilizing cFapydGuo:purine pairs induce local
melting of the duplex around the lesion site at 168C. Full
melting of the duplex would then explain the second higher
transition temperature. Alternatively, this second transition
could arise from the melting of an intramolecular hairpinlike
structure. Whatever the reason for the second melting curve
may be, it is clear that the cFapydGuo:purine-containing
duplexes show a first very low melting temperature indicative
of a strongly destabilized duplex structure. Most interesting
are, however, the dramatically different melting curves
obtained for the cFapydGuo:dA and 8-oxodGuo:dA
duplexes as depicted in Figure 1b. These curves clearly
show that in contrast to 8-oxodGuo, pairing of FapydGuo
with dA gives rise to strongly destabilized duplexes. We
therefore conclude that FapydGuo is unable to form proper
base pairs with dC and dA, which pair so well with 8-
oxodGuo. In contrast, the lesion FapydGuo seems to recog-
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