Changing selectivity of DNA oxidation from deoxyribose to guanine by ligand design and a new binuclear copper complex

Article abstract of DOI:10.1021/ja044209e

A dinuclear copper complex [Cu^II₂(PD-O)(H₂O)₂]³⁺ (1) (where PD-OH is a pyridylalkylamine containing binucleating ligand) promotes guanine oxidation in single-stranded DNA in the presence of 3-mercaptopropionic acid and dioxygen. This reaction is detected after subsequent piperidine treatment. Little spontaneous strand scission indicative of deoxyribose oxidation is observed in contrast to the results known for other copper complexes. Chemical characterization and nanospray ionization mass spectrometry analysis of oligodeoxynucleotides treated with 1 suggest conversion of guanine residues to their 2,6-diamino-5-formamidino-4-hydroxypyrimidine (+18 amu) and possibly 5,8-dihydroxy-7,8-dihydroguanine (+34 amu) derivatives. The selectivity toward nucleobase rather than deoxyribose oxidation is discussed in terms of the specific nature of the dicopper (hydro)peroxo species formed with the PD-OH ligand versus the intermediates formed in the presence of other binucleating ligands. Copyright

Full text of DOI:10.1021/ja044209e

Published on Web 12/23/2004
Changing Selectivity of DNA Oxidation from Deoxyribose to Guanine by
Ligand Design and a New Binuclear Copper Complex
Lei Li,† Kenneth D. Karlin,*,† and Steven E. Rokita*,‡
Department of Chemistry, The Johns Hopkins UniVersity, Baltimore, Maryland 21218, and Department of Chemistry
and Biochemistry, UniVersity of Maryland, College Park, Maryland 20742
Received September 23, 2004; E-mail: karlin@jhu.edu; rokita@umd.edu
Transition-metal complexes that cleave DNA have received
considerable attention in the past few years.1 One of the most
thoroughly studied is the copper bis(1,10-phenanthroline) complex,
which associates with the minor groove and cleaves duplex DNA
by direct abstraction of the deoxyribose H1′ hydrogen.1a,2 Recent
advances on this topic have provided important new insights into
the mechanism of DNA cleavage3 and inspired investigations on
many other copper complexes possessing DNA-cleaving reactivity.4
We previously reported that binuclear and trinuclear (but not
mononuclear) copper complexes utilizing pyridylalkylamine ligands
efficiently promote cleavage of DNA by oxidation of selected
deoxyribose moieties on single-stranded regions adjacent to junc-
tions of single- and double-stranded (ss/ds) DNA.5 A new dicopper-
(II) complex [CuII (PD′O)(H2O)2](ClO4)3‚2H2O (1) (Chart 1)
2
reported here acts in a contrasting manner by primarily promoting
nucleobase (vs deoxyribose) oxidation at unpaired guanine residues,
particularly at such junctions.
Figure 1. (A) Secondary structure of OD1/OD2 and OD3/OD4. (B)
Previous studies show that low temperature (organic solvent)
oxygenation of [CuI2(D1)]2+, a dicopper(I) analogue of 2 (Chart
1), leads to the formation a peroxo-dicopper(II) species.6 This
intermediate may be directly involved in oxidation of DNA, since
reduction of 2 and the presence of O2 are required for DNA strand
cleavage.5 The structure and reactivity of copper-dioxygen adducts
(such as peroxo dicopper(II), superoxo copper(II), bis-µ-oxo
dicopper(III)) strongly depend on the ligands and the resulting
coordination environment.7 Thus, we sought to manipulate the
possible intermediate responsible for DNA oxidation by employing
a binucleating phenolate containing chelate such as in 1 (Chart 1).
Related complexes form peroxo and/or hydroperoxo dicopper(II)
species, the latter of which may oxidize substrates via electrophilic
reactions.8 The binucleating ligand PD′OH forming a bis(aquo)
dicopper(II) complex 1 was consequently synthesized.9
Phosphoimage of a 20% polyacrylamide denaturing gel (7 M urea) of 100
nM 5′-32P-OD1+OD2 incubated in the presence and absence of 1 (100
µM) and MPA (5 mM) for 15 min in sodium phosphate (10 mM, pH 6.8)
at ambient temperature. Lanes 1, 5: OD1 alone. Lanes 2, 6: OD1 + OD2
and MPA. Lanes 3, 7: OD1+OD2 with 1. Lanes 4, 8: OD1 + OD2 with
1 and MPA. Lanes 5-8: treated with 0.2 M piperidine at 90 °C for 30
min.
Chart 1
The reactivity of 1 was first examined with the ss/ds-forming
oligodeoxynucleotides (OD1 + OD2) in the presence of excess
reductant 3-mercaptopropionic acid (MPA) under ambient condi-
tions (aerobic) and quenched by diethyl dithiocarbamic acid after
15 min.5b Subsequent treatment with piperidine revealed significant
and selective cleavage at G20 and G21 of OD1 at the junction, while
little reaction was apparent in the absence of this secondary
treatment (Figure 1, lane 8 vs 4). Cleavage at these G’s accounted
for ca. 70% of the total reaction, and no other G’s in ss or ds regions
reacted above a low background level (e.g., G23 and G15). Equivalent
specificity was also observed for G6 and G7 in OD2.9 This target
structure had been chosen initially based on the selectivity of the
previous multinuclear copper complexes for ss/ds junctions.5
However, when OD1 itself was treated under similar conditions,
all G’s reacted although their efficiency was lower than that for
G’s in a junction.9
The lack of direct strand cleavage of DNA suggested that the
reactive derivative of 1 selectively oxidized the nucleobase instead
of deoxyribose, the most common target of copper complexes.
Hydrogen atom abstraction from all positions of the deoxyribose
except for C1′ and C2′ yields at least some direct strand scission.3d,10
The C1′ product is labile under relatively mild conditions (NaOH,
0.1 M, 37 °C, 20 min).10 These conditions were not sufficient for
inducing scission in DNA after its reaction with 1. The nearly
complete lack of spontaneous strand scission is highly unusual, but
known in a few cases, for simple copper salts or complexes.11
8-Oxo-7,8-dihydro-2′-guanine (8-oxoG) is perhaps the most
frequent product of nucleobase oxidation in DNA.11 Although
8-oxoG is not piperidine labile, its low redox potential makes it
prone to further aerobic oxidation that ultimately leads to cleavage
under piperidine treatment.12 Such oxidation may be stimulated by
2- 13
IrCl6
, but this did not enhance the cleavage of DNA products
† The Johns Hopkins University.
‡ University of Maryland.
formed here by reaction of 1.9 Addition of 2-mercaptoethanol to a
9
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10.1021/ja044209e CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
piperidine treatment is known to act in a reciprocal manner by
preventing oxidation of 8-oxoG and strand scission.12a This modified
treatment also had no detectable effect on the yield of DNA
cleavage after reaction with 1, MPA, and O2.9 Thus, 8-oxoG is not
likely the product of oxidation under these conditions.
In summary, we have discovered a dicopper complex that
predominantly effects DNA base (rather than ribose) oxidation on
G residues in single-stranded regions of DNA. FAPy-G is one of
the major oxidation products derived from G. We hypothesize that
(i) the nature of the ligand-induced copper-dioxygen species and
(ii) copper-guanine binding18 are important elements in the
specificity of base recognition and oxidation. Further investigation
into the origins of these features are in progress.
Acknowledgment. We thank Dr. N. N. Murphy for helping with
the synthesis of the PD′OH ligand. This work was supported by
the NIH (GM28962 to K.D.K. and GM47531 to S.E.R.).
Supporting Information Available: Synthesis of the copper
complex (1) and PAGE analysis of DNA oxidation (PDF). This material
is available free of charge via the Internet at http://pubs.acs.org.
To provide further insight into the nucleobase (G) oxidation
chemistry, an oligodeoxynucleotide system (OD3 + OD4; Figure
1) of lower molecular weight than the original target was chosen
for analysis by mass spectrometry. Again, each strand contained
unpaired G’s for reaction with 1, and each demonstrated reactivity
similar to OD1 + OD2.9 Modification at ss G’s remained dominant
even after extended incubations (90 min, 65% yield) used to
maximize products for isolation and detection.9 Parent strands and
their derivatives were isolated from reverse-phase HPLC and
analyzed by nanospray ionization mass spectrometry (NSI/MS). For
OD3, major products of +18 amu and +34 amu were detected,9
and equivalent derivatives were detected for OD4. The species with
a +18 amu is consistent with formation of a 2,6-diamino-5-
formamidino-4-hydroxypyrimidine (FAPy-G) residue. Such a de-
rivative would explain the piperidine lability (see above). FAPy-G
is typically generated by hydroxyl radical addition followed by one-
electron reduction.11,14 Excess MPA likely facilitates the final
reduction step under the conditions used here.15 This product is
not consistent with generation of singlet oxygen (1O2) as proposed
for reaction of Cu(II) and H2O2.11d We only found one proposal in
the literature to explain the gain of + 34 amu.12b The suggested
product, 5,8-dihydroxy-7,8-dihydroguanine, is likely to be hydro-
lytically unstable and may rearrange to a more stable isomer.
The selectivity demonstrated by the copper complex 1 is atypical
at two levels. First, its preference for nucleobase rather than
deoxyribose oxidation is unlike most copper complexes known to
react with DNA. Although the copper-bound hydroxy radical
commonly proposed for reaction has the potential to add to guanine,
hydrogen abstraction of the deoxyribose is far more usual.10c,11a
Proximity and accessibility alone cannot explain the reaction
specificity because both 1 and 2 act on single-stranded regions that
should not restrict nucleotide access, and yet 1 primarily oxidizes
the nucleobase and 2 oxidizes the deoxyribose. Thorp has suggested
that systematic variation of ligand environment and accessibility
to a metal-oxo species may yield a series of oxidants selective for
either sugar or base.16 As mentioned above, [CuI2(D1)]2+ reacts with
O2 to produce a peroxo dicopper(II) complex,6 while the dicopper-
(I) analogue of 1 yields a hydroperoxo dicopper(II) complex.17
Theoretically, this latter species is a stronger (electrophilic) oxidant.8
Similar intermediates are expected to form during reaction with
DNA through initial MPA reduction of Cu(II) to Cu(I) followed
by reaction with ambient O2.5 Thus, the structural makeup or
specific nature of the dicopper-dioxygen intermediate instead of
its oxidizing power likely determines the type of DNA damage
produced.
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