Synthesis, oligonucleotide incorporation and base pair stability of 7-methyl-8-oxo-2′-deoxyguanosine

Article abstract of DOI:10.1039/b612597b

7-Methyl-8-oxo-2′-deoxyguanosine, an analogue of the abundant promutagen 8-oxo-2′-deoxyguanosine, was incorporated into oligonucleotides and tested for its stability in various base pairs. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b612597b

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis, oligonucleotide incorporation and base pair stability of
7-methyl-8-oxo-2ꢀ-deoxyguanosine†
Michelle L. Hamm* and Kelly Billig
Received (in Pittsburgh, PA, USA) 4th September 2006, Accepted 3rd October 2006
First published as an Advance Article on the web 13th October 2006
DOI: 10.1039/b612597b
7-Methyl-8-oxo-2ꢀ-deoxyguanosine, an analogue of the abun-
dant promutagen 8-oxo-2ꢀ-deoxyguanosine, was incorpo-
rated into oligonucleotides and tested for its stability in
various base pairs.
8-Oxo-2ꢀ-deoxyguanosine (OdG) is an abundant DNA lesion that
arises through exposure of dG to chemical carcinogens, radiation
and metabolic respiration.1 It is one of the most common damaged
nucleotides in mammalian cells,2 and has been linked to ageing3
as well as cancer.4 Previous studies have shown that OdG forms
stable base pairs with both dC and dA,5,6 and it can lead to
dG → T tranversions in vivo.7 Though OdG adopts an anti
conformation and utilizes classic Watson–Crick hydrogen bonds
when base pairing to dC,5 it adopts the syn conformation and uses
its Hoogsteen edge for hydrogen bonding when base pairing to
dA.6 OdG differs from dG at only the N7 and C8 positions, and
both these sites are key to its promiscuous base pairing. It has
been shown that the steric bulk of the oxygen at C8 destabilizes
the anti conformation,8 thereby destabilizing OdG(anti):dC base
pairs, while the N7-hydrogen can act as a hydrogen bond donor
Though the methylated product could be isolated in good yield,
and stabilize OdG(syn):dA mismatches. Due to these, and possibly
other reasons, OdG(anti):dC base pairs are only a bit more
stable that OdG(syn):dA base pairs,9 despite having an additional
hydrogen bond.
the material consistently decomposed during the oxidation step,
most likely due to depurination. Even after much investigation, no
suitable procedure could be found. Thus we decided to synthesize
the ribose derivative and then employ Barton–McCombie chem-
istry to deoxygenate at C2ꢀ. Since the standard protecting group
for the exocyclic amine of dG in DNA solid-phase synthesis is an
isobutyryl (iBu) group, we first synthesized the tetraisobutyrylated
rG derivative (1) through known procedures.13 The nucleoside
was then methylated at N7 with iodomethane before oxidation
at C8 with hydrogen peroxide in acetic acid to form N,2ꢀ,3ꢀ,5ꢀ-
tetraisobutyryl-7-methyl-8-oxoguanosine (3; Scheme 1). Selective
deprotection of the sugar alcohols with sodium methoxide before
selective protection of the 3ꢀ- and 5ꢀ-alcohols with a tetraisopropy-
ldisiloxy (TIPDS) group yielded compound 4. After derivatizing
the free 2ꢀ-hydroxyl with a phenoxythiocarbonyl group, it was
easily removed by reductive cleavage with tributyltin hydride and
AIBN to give the 2ꢀ-deoxy derivative 5. The TIPDS group was then
removed with 0.5 M TBAF in THF and the isobutyryl-protected
MdG derivative 6 was isolated. Finally, in order to prepare MdG
for chemical DNA synthesis, the 5ꢀ-hydroxyl was protected as a
dimethoxytrityl (DMTr) ether and the 3ꢀ-hydroxyl was activated
as a phosphoramidite.
In response to the abundance and promutagenicity of OdG, cells
have evolved repair enzymes that remove OdG from OdG:dC base
pairs.10 Since none of these enzymes are active with dG:dC base
pairs, the N7 and/or C8 positions must be an important element
for substrate recognition by these enzymes. Several crystal struc-
tures have provided evidence for a direct interaction between the
N7-hydrogen and the various enzymes,11 while biochemical studies
suggest the C8-oxygen may be important instead.10 To address
these questions and provide a means to alter the N7 position while
leaving the C8-oxygen unchanged, we set about synthesizing 7-
methyl-8-oxo-2ꢀ-deoxyguanosine (MdG) and incorporating it into
DNA.
Though 7-methyl-8-oxoriboguanosine has been reported
previously,12 the 2ꢀ-deoxy derivative (MdG) is not known. We
first attempted to synthesize MdG using a method similar to that
reported for 7-methyl-8-oxoriboguanosine by methylating a fully
protected dG derivative at N7 and then oxidizing at C8.
Standard synthesis and deprotection conditions were used
to construct an oligonucleotide 11 nucleotides long, with the
sequence 5ꢀ-dCCATCXCTACC-3ꢀ, where X is MdG (9a). After
purification by 20% PAGE and RP-HPLC, 9a was characterized
by MALDI-TOF mass spectrometry, as well as nuclease digest
experiments (see Supplementary information†). To complete the
Department of Chemistry, University of Richmond, Richmond, VA,
23173, USA. E-mail: mhamm@richmond.edu; Fax: +1 (804)287-1897.
Tel: +1 (804)287-6327
† Electronic supplementary information (ESI) available: Synthesis, purifi-
cation and characterization of 2–8, 9a and 10. Procedures and full or raw
data for oligonucleotide digestion and analysis, and melting and NMR
studies. See DOI: 10.1039/b612597b
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Scheme 1 Synthesis of the phosphoramidite derivative of MdG ready for solid-phase synthesis. Reagents and conditions: (i) CH3I, DMF, 37 ◦C, overnight;
(ii) 30% H2O2, AcOH, 37 ◦C, 3.25 h; (iii) 0.2 M NaOH in 65 : 35 pyridine–MeOH, 0 ◦C, 30 min; (iv) TIPDS-Cl, pyridine, rt, 1 h; (v) ClC(S)OPh, CH2Cl2,
rt, 2 h; (vi) Bu3SnH, AIBN, toluene, 70 ◦C, 1.5 h; (vii) 0.5 M TBAF in THF, rt, 2.5 h; (viii) DMTr-Cl, DMAP, pyridine, rt, 1.25 h; (ix) (iPr)2NEt,
Me-imidazole, ClP(N(iPr)2)OCH2CH2CN, CH2Cl2, rt, 30 min.
series, oligonucleotides where X = dG (9b) and OdG (9c) were
purchased and purified as well. All three oligonucleotides were
then paired with complementary oligonucleotides that contained
one of the four naturally occuring deoxynucleotides opposite the X
position, and tested for their stability in melting studies (Table 1).14
Since all duplexes were melted under the same conditions and they
all contained the same sequence except at the one base pair, the
stabilities of the varying base pairs can be directly compared.
Similar to previous results, OdG:dC and OdG:dA base pairs
were less and more stable, respectively, than dG:dC and dG:dA
base pairs. Interestingly, MdG:dC and OdG:dC base pairs were
of similar stability, indicating the absence of the N7-hydrogen
and the presence of the N7-methyl are both unimportant to the
stability of this base pair. Not surprisingly, MdG:dA base pairs
were significantly less stable than OdG:dA base pairs. This can
easily be explained not only by the absence of an N7-hydrogen,
but also by the presence of the N7-methyl, which could be in
steric clash with the N1 of dA if MdG is in the syn conformation.
Additionally, MdG:dA, and MdG:T and OdG:T base pairs were
all less stable than dG:dA and dG:T base pairs, respectively. Since
dG often adopts the anti conformation when base pairing to dA
and T (as in dG:dC base pairs; Scheme 2),15 the steric bulk of
the C8-oxygen in MdG and OdG would also destabilize these
mismatches if they are also in the anti conformation. Finally, OdG
and MdG formed less stable base pairs with dG than did dG itself.
Even though dG:dG base pairs usually involve one dG in the
anti conformation and the other dG in the syn conformation,16
neither of these options are suitable for OdG and MdG, since the
steric clash of the C8-oxygen would destabilize the base pair if
they adopted the anti conformation, while neither has the strong
N7 hydrogen bond accepting ability required when in the syn
conformation.
Table 1 Melting temperatures (Tm/◦C) of DNA duplexesa
Y = dC
Y = dA
Y = T
Y = dG
X = dG
57.5 0.5
52.7 0.5
53.3 0.2
43.2 0.2
48.0 0.3
39.0 0.2
46.3 0.4
41.5 0.2
41.2 0.6
45.1 0.2
40.9 0.5
38.3 0.6
X = OdG
X = MdG
a Average Tm
standard deviation was calculated from three or more
melting experiments.
Scheme 2 Common structures for base pair mismatches with dG.
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To further confirm that the C8-oxygen destabilizes the anti
conformation in MdG, we synthesized the MdG mononucleo-
side from its isobutyrylated derivative (Scheme 3). Deprotection
through treatment with methoxide, followed by purification
through a reverse-phase C18 column yielded the pure monomer 10.
It is known that an anti-to-syn nucleoside conformational change
results in strong downfield and upfield shifts in the H2ꢀ and C2ꢀ
signals, respectively.17 Such shifts were observed for MdG when
compared to dG (see Supplementary information†), consistent
with MdG prefering the syn conformation. These results further
the argument that the presence of the C8-oxygen destabilizes the
anti conformation, thereby destabilizing base pairs that contain
MdG in the anti conformation.
of this work. M.L.H. is a Camille and Henry Dreyfus Foundation
Start-up Awardee.
Notes and references
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14 Melting temerature data were obtained using 1 mL solutions that
contained 5 lM duplex, 1 M NaCl, 0.1 mM EDTA and 100 mM
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at 90 ◦C, before cooling to 4 ◦C over at least 1 h. The absorbance at
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Scheme 3 Synthesis of the MdG monomer. Reagents and conditions:
(i) 0.3 M NaOMe in HOMe, 40 ◦C, 5 h.
In conclusion, we have developed an efficient route toward the
incorporation of the OdG analogue MdG into oligonucleotides.
It should be noted that though this adduct may be produced
naturally in cells, it is highly unlikely due to the already low
abundance of either OdG or 7-methyl-2ꢀ-deoxyguanosine. Still,
MdG is of great importance since it has the potential to help
elucidate the role of the N7-hydrogen in the biological activities
of OdG. We are currently testing the activity of various repair
enzymes with MdG in order to better understand their modes of
substrate recognition.
Acknowledgements
The authors would like to thank Tim Smith and Bojan Dragulev
for help with mass spectrometry and DNA synthesis, respectively.
This work was partially supported by the NSF CAREER (CHE-
0239664) and MRI (CHE-0116492 and CHE-0320669) programs.
Acknowledgement is made to the donors of the American
Chemical Society Petroleum Research Fund for partial support
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4070 | Org. Biomol. Chem., 2006, 4, 4068–4070
This journal is
The Royal Society of Chemistry 2006
©