reactive and may be the primary cause for oxidative DNA
damage by chromium.17 Our past work has shown that, when
Cr(VI) is reduced by ascorbic acid in the presence of duplex
DNA, preferential oxidation of guanine will occur to generate
8-oxoG and Sp.18 Extension of this work with Cr(VI)-treated
Escherichia coli has identified the formation of Sp and
8-oxoG in cellular DNA.19 Our studies have shown Sp to
be the dominant lesion formed, and we propose that the
cellular toxicity of chromium is partially due to the ability
of high-valent Cr complexes to form the Sp lesion from the
further oxidation of 8-oxoG.
Figure 2. Typical reverse phase chromatogram showing the
separation of products of the oxidation of acyl-8-oxoG by Cr(V)-
salen at pH 7.
N,N′-Ethylenebis(salicylideneanimato)oxochromium(V)
(Cr(V)-salen) 1 and bis(2-ethyl-2-hydroxybutyrato)oxochro-
mium(V) (Cr(V)-ehba) 2 (Figure 1) are two models of
the spiro ring system.21 It was unequivocally identified by
its characteristic UV absorption spectrum, showing the 230
nm shoulder (Figure 2), and by its signature mass spectrum,
showing the M + H peak at 442 amu, the M + Na peak at
464 amu, and a fragment B + H2 at 184 amu generated by
cleavage of the glycosidic bond.
The oxidation of 8-oxoG to Sp is a two-electron process.
The Cr(V)-salen and Cr(V)-ehba complexes are thought
to be two-electron oxidants,22 where the axial oxygen on the
Cr(V) complex is directly transferred to 8-oxoG, resulting
in an immediate two-electron oxidation species that forms
Sp while reducing Cr(V) to Cr(III). This mechanism implies
that a 1:1 stoichiometry of Cr(V) to 8-oxoG is required in
the oxidation reaction.
To determine if the oxidation mechanism involved an oxo-
atom transfer mechanism, acyl-8-oxoG was oxidized with
various stoichiometric equivalents of Cr(V)-salen or Cr(V)-
ehba. The data obtained were compared to the oxidation of
acyl-8-oxoG with IrCl62-, a reaction shown to occur by
sequential one-electron transfer processes.12 The increase in
product 4 and decrease in reactant 3 was monitored by HPLC
(Figure 3A). One equivalent of Cr(V)-ehba or Cr(V)-salen
was sufficient to oxidize nearly all the acyl-8-oxoG to acyl-
Figure 1. (A) Cr(V) complexes used to model cellular high-valent
Cr complexes. (B) Oxidation of 8-oxoG by Cr(V).
cellular high-valent Cr.16,20 Due to the mixed nitrogen/oxygen
ligand chelation, the Cr(V)-salen complex is proposed to
mimic chromium-peptide interactions, while the Cr(V)-
ehba complex mimics the chromium-ascorbic acid complex.
Both complexes have been shown to preferentially oxidize
duplex DNA at guanine and preferentially oxidize 8-oxoG
in DNA containing an 8-oxoG lesion.6,20 Here, we report on
the mechanism of formation of the major product of 2′,3′,5′-
triacetoxy-8-oxo-2′-deoxyguanosine 3 (acyl-8-oxoG) oxida-
tion by Cr(V)-salen 1 or Cr(V)-ehba 2.
The oxidation of a 1.5 mM solution of 3 in aqueous
phosphate buffer (75 mM, pH 7) at 37 °C by 0.75 mM
Cr(V)-salen 1 or Cr(V)-ehba 2 leads to the formation of
only one stable oxidation product, 2′,3′,5′-triacetoxyspiroimi-
nodihydantoin 4 (acyl-Sp). Acyl-Sp was identified by HPLC
(Figure 2) eluting at 10 min and was observed as a double
peak from the mixture of epimers formed on generation of
2-
Sp, whereas two equivalents of IrCl6 was required for a
complete reaction. These data imply that the Cr(V) com-
plexes do indeed undergo a two-electron transfer process
under these conditions.
The previous mechanism for the formation of Sp using
one-electron oxidants via an electron abstraction mechanism12
identified 5-hydroxy-8-oxoG, 5, as an intermediate. At pH
7, intermediate 5 undergoes an acyl shift to form the Sp
derivative 4. We considered, therefore, the possibility that
the Cr(V) oxidants were directly forming 4 by two-electron
oxidation through the transfer of the axial oxygen, but the
possibility of two sequential single electron abstractions could
not be ruled out. To resolve this, we oxidized 3 in H218O
using the high-valent Cr(V) species 1 and 2 or Ir(IV). If oxo-
atom transfer were occurring, a 16O-Sp-containing product,
m/z ) 300 for the nonacylated species, would form since
the donated oxygen would be supplied directly from the axial
16O oxygen of the Cr complex. As previously demon-
strated,12,18 oxidation occurring by two single electron
(15) Rossi, S. C.; Gorman, N.; Wetterhahn, K. E. Chem. Res. Toxicol.
1988, 1, 101-107.
(16) Liebross, R. H.; Wetterhahn, K. E. Chem. Res. Toxicol. 1990, 3,
401-403.
(17) Sugden, K. D.; Wetterhahn, K. E. Inorg. Chem. 1996, 35, 651-
657.
(18) Slade, P. G.; Hailer, M. K.; Martin, B. D.; Sugden, K. D. Chem.
Res. Toxicol. 2005, 18, 1140-1149.
(19) Slade, P. G.; Hailer, M. K.; Martin, B.; Sugden, K. D. Chem. Res.
Toxicol. 2005, 18, 1378-1383.
(20) Sugden, K. D.; Martin, B. D. EnViron. Health Perspect. 2002, 110,
725-728.
(21) For analytical HPLC-DAD and HPLC-ESI-MS, the system used as
previously described.18
(22) Holm, R. H. Chem. ReV. 1987, 87, 1401-1449.
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Org. Lett., Vol. 9, No. 22, 2007