Published on Web 09/18/2007
Stereospecific Synthesis and Characterization of
Oligodeoxyribonucleotides Containing an
N2-(1-Carboxyethyl)-2′-deoxyguanosine
Huachuan Cao, Yong Jiang, and Yinsheng Wang*
Contribution from the Department of Chemistry, UniVersity of California,
RiVerside, California 92521-0403
Received March 26, 2007; E-mail: yinsheng.wang@ucr.edu
Abstract: Methylglyoxal is a highly reactive R-ketoaldehyde that is produced endogenously and present in
the environment and foods. It can modify DNA and proteins to form advanced glycation end products (AGEs).
Emerging evidence has shown that N2-(1-carboxyethyl)-2′-deoxyguanosine (N2-CEdG) is a major marker
for AGE-linked DNA adducts. Here, we report, for the first time, the preparation of oligodeoxyribonucleotides
(ODNs) containing individual diastereomers of N2-CEdG via a postoligomerization synthesis method, which
provided authentic substrates for examining the replication and repair of this lesion. In addition,
thermodynamic parameters derived from melting temperature data revealed that the two diastereomers of
N2-CEdG destabilized significantly the double helix as represented by a 4 kcal/mol increase in Gibbs free
energy for duplex formation at 25 °C. Primer extension assay results demonstrated that both diastereomers
of N2-CEdG could block considerably the replication synthesis mediated by the exonuclease-free Klenow
fragment of Escherichia coli DNA polymerase I. Strikingly, the polymerase incorporated incorrect nucleotides,
dGMP and dAMP, opposite the lesion more preferentially than the correct nucleotide, dCMP.
substrates to form advanced glycation end products (AGEs).12,13
If remain unrepaired, those AGEs could eventually lead to the
Introduction
Humans are exposed to methylglyoxal (MG), a highly reactive
R-ketoaldehyde, from a variety of sources. General exogenous
sources of MG include cigarette smoke,1 food, and beverages
such as soy sauce, coffee, and whiskey.2,3 On the other hand,
the fragmentation of triose phosphate during glycolysis, me-
tabolism of acetone, and catabolism of aminoacetone all
contribute to the endogenous formation of MG.4,5 Many factors,
including aging, hyperglycemia, inflammation, oxidative stress,
and uremia, could enhance the accumulation of MG in ViVo.6
In this respect, as high as 310 µM of methylglyoxal was detected
in Chinese hamster ovary cells.7 In addition, a previous clinical
study indicated that the median level of MG increased by several
fold in blood samples from type I and II diabetic patients, and
the concentration of MG correlates positively with the duration
and development of type I diabetes.8
inhibition of enzyme activity,14 transcription activation,15 and
apoptosis.16
Several MG adducts of DNA have been previously reported,
and guanine is the predominant modification site induced by
MG when calf thymus DNA was used as substrate.13,17,18 In an
earlier study, N2-(1-carboxyethyl)-9-methylguanine was formed
from the incubation of 9-methylguanine with glucose or MG;
thus it was postulated that N2-(1-carboxyethyl)-2′-deoxygua-
nosine (N2-CEdG) could be induced in DNA by MG.19 Indeed
Frischmann et al.13 showed recently that N2-CEdG was the stable
adduct formed in calf thymus DNA upon prolonged exposure
to MG at physiological concentration and temperature. These
authors further proposed that the N2-CEdG could be produced
from either direct coupling of 2′-deoxyguanosine (dG) with MG
or from the conversion of 1,N2-(1,2-dihydroxy-2-methyl)ethano-
2′-deoxyguanosine (cMG-dG) (Scheme 1).
As a reactive dicarbonyl compound, MG is involved in
numerous pathological processes in ViVo by binding irreversibly
to proteins, primarily on arginine residues,9-11 DNA, and other
(11) Oya, T.; Hattori, N.; Mizuno, Y.; Miyata, S.; Maeda, S.; Osawa, T.; Uchida,
K. J. Biol. Chem. 1999, 274, 18492-18502.
(1) Moreetesta, P.; Saintjalm, Y. J. Chromatogr. 1981, 217, 197-208.
(2) Nagao, M.; Fujita, Y.; Wakabayashi, K.; Nukaya, H.; Kosuge, T.; Sugimura,
T. EnViron. Health Perspect. 1986, 67, 89-91.
(3) Nagao, M.; Fujita, Y.; Sugimura, T.; Kosuge, T. IARC Sci. Publ. 1986,
283-291.
(4) Kalapos, M. P. Toxicol. Lett. 1999, 110, 145-175.
(5) Phillips, S. A.; Thornalley, P. J. Eur. J. Biochem. 1993, 212, 101-105.
(6) Ramasamy, R.; Yan, S. F.; Schmidt, A. M. Cell 2006, 124, 258-260.
(7) Chaplen, F. W. R.; Fahl, W. E.; Cameron, D. C. Proc. Natl. Acad. Sci.
U.S.A. 1998, 95, 5533-5538.
(8) McLellan, A. C.; Thornalley, P. J.; Benn, J.; Sonksen, P. H. Clin. Sci. 1994,
87, 21-29.
(9) Ahmed, N.; Thornalley, P. J. Biochem. J. 2002, 364, 15-24.
(10) Gao, Y.; Wang, Y. S. Biochemistry 2006, 45, 15654-15660.
(12) Lo, T. W. C.; Westwood, M. E.; McLellan, A. C.; Selwood, T.; Thornalley,
P. J. J. Biol. Chem. 1994, 269, 32299-32305.
(13) Frischmann, M.; Bidmon, C.; Angerer, J.; Pischetsrieder, M. Chem. Res.
Toxicol. 2005, 18, 1586-1592.
(14) Murata-Kamiya, N.; Kamiya, H. Nucleic Acids Res. 2001, 29, 3433-3438.
(15) Maeta, K.; Izawa, S.; Okazaki, S.; Kuge, S.; Inoue, Y. Mol. Cell. Biol.
2004, 24, 8753-8764.
(16) Fukunaga, M.; Miyata, S.; Liu, B. F.; Miyazaki, H.; Hirota, Y.; Higo, S.;
Hamada, Y.; Ueyama, S.; Kasuga, M. Biochem. Biophys. Res. Commun.
2004, 320, 689-695.
(17) Shapiro, R.; Cohen, B. I.; Shiuey, S. J.; Maurer, H. Biochemistry 1969, 8,
238-245.
(18) Krymkiew, N. FEBS Lett. 1973, 29, 51-54.
(19) Papoulis, A.; Alabed, Y.; Bucala, R. Biochemistry 1995, 34, 648-655.
9
10.1021/ja072130e CCC: $37.00 © 2007 American Chemical Society
J. AM. CHEM. SOC. 2007, 129, 12123-12130
12123