A Synthetic Nucleoside Probe that Discerns a DNA Adduct from Unmodified DNA
Jiachang Gong and Shana J. Sturla*
Department of Medicinal Chemistry and The Cancer Center, UniVersity of Minnesota, Minneapolis, Minnesota 55455
Received January 30, 2007; E-mail: firstname.lastname@example.org
Biologically reactive chemicals alkylate DNA and induce
structural modifications in the form of covalent adducts.1Certain
bulky DNA adducts can persist, escape repair, and serve as
templates for polymerase-mediated DNA synthesis, resulting in
mutation and cancer.2Correlating chemical structures and quantita-
tive levels of adducts with toxicity is central to understanding
chemical mechanisms of carcinogenesis for specific agents. Major
challenges include that DNA adducts are formed at exceedingly
low levels, adduct mixtures are often formed, and minor lesions
may have greater biological impact than more abundant products.2
New molecular approaches for addressing specific low-abundance
adducts are needed, and we describe here the first example of a
synthetic nucleoside that may serve as the chemical basis for a probe
of a bulky carcinogen-DNA adduct.
Dozens of thermodynamically stable synthetic base pairs have been
reported3and continue to emerge as powerful tools in areas such as
polymerase fidelity,4DNA helix stability,5nucleic acids with novel
functionality,6and expanded genetic systems,3to cite selected exam-
ples. Recently, Hirao and co-workers successfully have amplified
an entirely synthetic base pair.7Amplified in a polymerase-mediated
process or used in hybridization-based strategies, synthetic nucleo-
sides might act as probes of DNA damage, but to our knowledge,
no examples of synthetic nucleosides that pair selectively with an
adduct generated in a natural physiological system are known.
O6-Benzyldeoxyguanosine (1, O6-BnG; Figure 1) is a bulky DNA
adduct chosen for analysis because of its prominent role in nucleic
acid chemistry and biology and the high frequency of O6-
alkylguanine lesions.8,9This adduct results naturally from exposure
to environmental carcinogens8a,band is highly mutagenic, causing
G to C and G to T transversion, and G to A transition mutations.8c,d
O6-Alkylguanine adducts have altered hydrogen-bonding capacity,
increased size, and decreased hydrophilicity relative to G (Figure 1).
On the basis of molecular modeling studies,10we anticipated that
a diaminonaphthyl-derived nucleoside (2, dNap; Figure 1) would
possess a hydrogen-bonding capacity complementary to O6-BnG
and favorable π-π stacking and hydrophobic interactions between
the benzyl moiety of O6-BnG and the naphthyl moiety of dNap 2.
To evaluate the O6-BnG:dNap base pair in duplex DNA, we
prepared a series of oligonucleotides containing selected combina-
tions of DNA adduct, synthetic nucleoside, and/or natural bases.
Nucleoside 2 was synthesized from diaminonaphthalene 3 (Scheme
1). Treatment of 3 with ethyl chloroformate produced perimidinone
4 (70% yield), which was coupled with bistoluoyl chloroglycoside
to yield the ?-isomer of 5 as the major product. Deprotection, 5′-
tritylation, and conversion to the 3′-phosphoramidite 6, required
for oligonucleotide synthesis, were achieved in 50% yield overall.
Duplex DNA stability was determined by thermal denaturation
of synthetic oligonucleotides. Melting temperatures (Tm) were mea-
sured for complementary sequences 5′-TTGTCGGTATAXC GG-
3′ and 5′-CCGYTATACCGACAA-3′ with varying bases incorpo-
rated at positions X and Y. The results indicate that O6-BnG:dNap
is markedly stable (8.0 µM) with a Tmvalue one degree lower than
that of the natural dG:dC pair (Figure 2, D1 Tm) 60.3 vs D6 Tm
) 59.3). Sequence D5 represents a situation in which natural DNA
is damaged, giving rise to the O6-BnG adduct and a diminished
thermal stability. Further, the adduct:probe pair Tmwas compared
to O6-BnG paired with canonical bases. These combinations have
diminished stabilities relative to the synthetic pair by 5.0, 6.6, 4.7,
and 5.9 °C for dG, dA, dC, and dT, respectively (Figure 2). Simi-
larly, for dNap paired opposite the natural bases, Tmdiminished to
55.3, 54.8, 52.5, and 52.5 °C for dG, dA, dC, and dT, respectively.
These data are comparable to optimized synthetic base pairs, in
which approximate ranges of 4-9 °C in Tm depressions are
considered highly stable and orthogonal systems.3eTmvalues for
point mutations in D1, which reflect the selectivity of natural base
pairs, decrease by an estimated average of 9 °C.11
Figure 1. Schematic representation of base-pair interactions for a standard
G:C pair, alkylation-damaged O6-BnG 1:C pair and proposed adduct:probe
combination (dR ) deoxyribose).
aReagents and conditions: (a) ethyl chloroformate, THF; (b) bistoluoyl
chloroglycoside, NaH/THF; (c) NaOMe/methanol; (d) 4,4′-dimethoxytrityl
chloride, pyridine; (e) N,N′-diisopropyl-2-O-cyanoethyl phosphoramidic
chloride, Et3N, CH2Cl2.
Figure 2. Thermal stabilities of natural, damaged, and dNap DNA.
Published on Web 04/03/2007
4882 9 J. AM. CHEM. SOC. 2007, 129, 4882-4883
10.1021/ja070688g CCC: $37.00 © 2007 American Chemical Society