of 25
Using Metal Complex Reduced States to Monitor the Oxidation
of DNA
Eric D. Olmon
,
Michael G. Hill
, and
Jacqueline K. Barton
†,*
Division of Chemistry and Chemical Engineering, California Institute of Technology Pasadena,
California 91125, USA
Department of Chemistry, Occidental College, Los Angeles, California 90041, USA
Abstract
Metallointercalating photooxidants interact intimately with the base stack of double-stranded DNA
and exhibit rich photophysical and electrochemical properties, making them ideal probes for the
study of DNA-mediated charge transport (CT). The complexes [Rh(phi)
2
(bpy
)]
3+
(phi = 9,10-
phenanthrenequinone diimine; bpy
= 4-methyl-4
-(butyric acid)-2,2
-bipyridine), [Ir(ppy)
2
(dppz
)]
+
(ppy = 2-phenylpyridine; dppz
= 6-(dipyrido[3,2-a:2
,3
-c]phenazin-11-yl)hex-5-ynoic acid),
and [Re(CO)
3
(dppz)(py
)]
+
(dppz = dipyrido[2,3-a:2
,3
-c]phenazine; py
= 3-(pyridin-4-yl)-
propanoic acid) were each covalently tethered to DNA in order to compare their photooxidation
efficiencies. Biochemical studies show that upon irradiation, the three complexes oxidize guanine
by long-range DNA-mediated CT with the efficiency: Rh > Re > Ir. Comparison of spectra
obtained by spectroelectrochemistry after bulk reduction of the free metal complexes with those
obtained by transient absorption (TA) spectroscopy of the conjugates suggests that the reduced
metal states form following excitation of the conjugates at 355 nm. Electrochemical experiments
and kinetic analysis of the TA decays indicate that the thermodynamic driving force for CT,
variations in the efficiency of back electron transfer, and coupling to DNA are the primary factors
responsible for the trend observed in the guanine oxidation yield of the three complexes.
INTRODUCTION
Cellular DNA is continually under the threat of oxidation from a host of sources.
1–4
Left
unrepaired, oxidative damage to DNA leads to health problems, including cancer.
5–7
In
order to improve our understanding of the chemical mechanisms underlying oxidative
damage, as well as the biological factors affecting the prevalence, detection, and repair of
such damage, it is necessary to utilize a wide variety of chemical and biological tools and
techniques.
One especially useful tool for the study of oxidative damage in DNA is DNA-mediated
charge transport (CT). Due to orbital overlap between the
π
systems of neighboring
nucleobases, DNA can serve as a bridge in long-range electron transfer (ET) reactions.
Unlike photocleavage mechanisms, many of which result in the formation of nonspecific
damage by reactive oxygen species,
8–10
or photoligation mechanisms, which lead to the
formation of unnatural adducts between metal complexes and DNA,
11
DNA-mediated CT
results in preferential damage at sites of low oxidation potential. Oxidative events at low
potential guanine sites (
E
°[G
•+
/G] = 1.29 V vs. NHE)
12
can be initiated by many different
*
To whom correspondence should be addressed. jkbarton@caltech.edu.
Supporting Information.
Cyclic voltammetry traces of
Re
and Re-OEt in acetonitrile, accumulation of guanine damage with
irradiation. This material is available free of charge via the internet at http://pubs.acs.org.
NIH Public Access
Author Manuscript
Inorg Chem
. Author manuscript; available in PMC 2012 December 5.
Published in final edited form as:
Inorg Chem
. 2011 December 5; 50(23): 12034–12044. doi:10.1021/ic201511y.
NIH-PA Author Manuscript
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NIH-PA Author Manuscript
DNA-bound oxidants, including organic molecules, transition metal complexes, and DNA
base analogues,
13–18
allowing for the study of DNA oxidation in a wide variety of
environments and sequence contexts. Additionally, oxidative probes are capable of inducing
damage in regions far from the site of charge injection. In solution studies, damage at
guanine sites was observed almost 200 Å away from a DNA-bound oxidant.
19
Recently, our
laboratory observed the propagation of robust redox signals over a distance of 100 base
pairs, or 340 Å, in DNA monolayers on gold electrodes.
20
DNA CT may fulfill biological
roles as well. The observed funneling of oxidative damage to regions of mitochondrial DNA
that contain genes necessary for replication may serve as a check against the propagation of
damaged genetic material in situations of high oxidative stress.
21
DNA CT also may be
involved in other capacities within the cell,
22
for example, to activate transcription
23,24
and
to perform long-range signaling.
25
In order to study such reactions in the laboratory, it is necessary to have a convenient
method for initiating DNA CT reactions.
26
Transition metal complexes have proven
especially amenable for use as oxidants in the study of DNA damage due to their synthetic
versatility and the ability to tune their redox properties. In addition, an appropriate ligand set
enables metal complexes to interact strongly with DNA through intercalative binding,
allowing for the initiation of long-range DNA-mediated oxidation by optical excitation of
the bound complex. Complexes of the type [Rh(phi)
2
(L)]
3+
(phi = 9,10-
phenanthrenequinone diimine), where L = bpy (2,2
-bipyridine) or phen (phenanthroline),
are especially strong photooxidants. These complexes, which bind DNA through
intercalation of the phi ligand, were used to establish the ability of DNA to propagate
charge.
27
Photoexcitation of DNA-bound [Rh(phi)
2
(L)]
3+
at 365 nm leads to injection of a
positive charge into the DNA base stack, which then equilibrates at sites of low redox
potential (guanine and guanine repeats).
19,28
Iridium complexes have also been used to
initiate DNA-mediated CT processes. The complex [Ir(ppy)
2
(dppz)]
+
(ppy = 2-
phenylpyridine; dppz = dipyrido[2,3-a:2
,3
-c]phenazine) intercalates into DNA via the dppz
ligand. Interestingly, from the excited state, the complex is a strong enough reductant and
oxidant to promote both the reduction and the oxidation of DNA.
29
This remarkable ability
has enabled characterization of DNA-mediated electron transfer and DNA-mediated hole
transfer in identical sequence contexts, showing that both have a shallow distance
dependence.
30,31
Tricarbonyl rhenium complexes are of interest due to the strong infrared
absorption of the carbonyl ligands. Excitation and reduction of such complexes can be
followed temporally by observing dynamic changes in the stretching frequencies of the
carbonyl ligands.
32–34
In addition, complexes such as [Re(CO)
3
(dppz)(L)]
n+
act as “light
switches,”
35
luminescing only when bound to DNA.
36–39
Such interesting photophysical
properties provide additional means of monitoring DNA CT events.
Due to the large number of factors that affect the relative efficiency of DNA CT, such as
DNA binding strengths, redox properties, and photophysical behavior of various metal
complexes, it is necessary to compare DNA oxidants in identical environments. In the
present study, we have examined the ability of three metal complexes to report on DNA-
mediated oxidation events through the appearance of their reduced states. We have focused
on investigation of the reduced states of [Rh(phi)
2
(bpy
)]
3+
, [Ir(ppy)
2
(dppz
)]
+
, and
[Re(CO)
3
(dppz)(py
)]
+
[
Rh
,
Ir
, and
Re
, respectively; bpy
= 4-methyl-4
-(butyric acid)-2,2
-
bipyridine; dppz
= 6-(dipyrido[3,2-a:2
,3
-c]phenazin-11-yl)hex-5-ynoic acid; py
= 3-
(pyridin-4-yl)-propanoic acid] and their DNA-conjugates (
Rh-DNA
,
Ir-DNA
, and
Re-
DNA
) in aqueous and organic solutions, as well as their efficiencies of DNA
photooxidation. The structures of the complexes and conjugates are shown in Figure 1. We
have used steady-state spectroelectrochemistry and nanosecond transient absorption (TA)
spectroscopy to record the electronic spectra of the reduced states of the metal complexes
and the charge transfer products of the metal-DNA conjugates, respectively. In addition, we
Olmon et al.
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. Author manuscript; available in PMC 2012 December 5.
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have compared these spectral profiles with the redox properties and efficiency of DNA
photooxidation of the three complexes.
EXPERIMENTAL SECTION
Materials
Unless indicated otherwise, all reagents and solvents were of reagent grade or better and
were used as received without further purification. All reagents for DNA synthesis were
purchased from Glen Research (Sterling, VA). The complexes
Ir
and
Rh
were laboratory
stocks and had been prepared from published protocols.
29,40
Synthesis of Metal Complexes
The synthesis of [
fac
-Re(CO)
3
(dppz)(py
)][Cl] closely followed the procedure of Stoeffler,
et al.
37
A mixture of 253 mg (0.7 mmol) Re(CO)
5
Cl and 147 mg (0.7 mmol) 1,10-
phenanthroline-5,6-dione in 7 mL toluene was refluxed (110 °C) for 4.5 h. The crude solid
product was collected by suction filtration, purified by silica gel using THF as an eluent, and
dried under vacuum to yield Re(CO)
3
Cl(1,10-phenanthroline-5,6-dione) as an orange
microcrystalline solid. Re(CO)
3
Cl(dppz) was formed by heating 160 mg (0.31 mmol)
Re(CO)
3
Cl(1,10-phenanthroline-5,6-dione) in 15 mL EtOH to reflux (85 °C), adding 55 mg
(0.6 mmol)
o
-phenylenediamine, and refluxing the mixture for 1 h. The yellow-ochre solid
product was collected by suction filtration.
1
H NMR (300 MHz) in DMSO indicated the
presence of the dppz ligand:
δ
8.22 (q, 2H), 8.31 (m, 2H), 8.55 (q, 2H), 9.58 (d, 2H), 9.88 (d,
2H). The desired product was obtained following substitution for the Cl ligand. A
suspension of 160 mg (0.27 mmol) Re(CO)
3
Cl(dppz) was heated under Ar to 50 °C in 25
mL dry DMF. After addition of 280 mg (1.1 mmol) AgPF
6
, the reaction mixture was heated
at 50 °C for 5 min., then 250 mg (1.7 mmol) py
was added and the mixture was refluxed at
70 °C under Ar for 6 h. The reaction was cooled, and the AgCl precipitate was removed by
gravity filtration, yielding an orange-yellow solution. The crude product was purified by
silica gel using 5% methanol in chloroform as the eluent, and then dried under vacuum to
yield [Re(CO)
3
(dppz)(py
)](PF
6
). The PF
6
counter ion was exchanged for chloride using
Sephadex QAE A-25 anion exchange resin, and the resulting solution was concentrated
using a C18 Sep-Pak to yield
fac
-[Re(CO)
3
(dppz)(py
)]Cl as a bright yellow solid.
1
H NMR
(PF
6
salt, 300 MHz, CD
3
CN):
δ
9.79 (dd, 2H), 9.65 (dd, 2H), 8.37 (dd, 2H), 8.23 (m, 4H),
8.09 (dd, 2H), 7.13 (d, 2H), 2.73 (t, 2H), 2.44 (t, 2H).
13
C NMR (PF
6
salt, 300 MHz,
CD
3
CN):
δ
155.3, 155.0, 151.3, 149.0, 142.4, 136.5, 132.4, 129.3, 128.2, 126.3, 32.3, 28.9.
ESI: calcd 703.7 for C
29
H
19
N
5
O
5
Re (M
+
), found 703.9.
The related ethyl ester was prepared in the same way following the Fischer esterification of
py
.
1
H NMR (PF
6
salt, 300 MHz, CD
3
CN):
δ
9.90 (dd, 2H), 9.65 (dd, 2H), 8.45 (dd, 2H),
8.26 (dd, 2H), 8.20 (dd, 2H), 8.13 (dd, 2H), 7.09 (d, 2H), 3.88 (q, 2H), 2.72 (t, 2H), 2.41 (t,
2H), 0.99 (t, 3H). ESI: calcd 731.8 for C
31
H
23
N
5
O
5
Re (M
+
), found 732.0.
DNA Synthesis and Modification
Oligonucleotides were prepared using standard solid-phase phosphoramidite chemistry on
an Applied Biosystems 3400 DNA synthesizer. Covalent tethers were appended to the 5
-
ends of resin-bound oligonucleotides in two ways. For the
Ir-DNA
conjugate, an amino-
terminated C
6
-alkyl phosphoramidite was added in the last step of the automated synthesis;
for the
Rh-
and
Re-DNA
conjugates, a diaminononane linker was added as previously
described.
41
Agitation of the amine modified strands in the presence of metal complex,
O
-
(benzotriazol-1-yl)-
N,N,N
,
N
-tetramethyluronium hexafluorophosphate (HBTU), 1-
hydroxybenzotriazole hydrate (HOBT), and diisopropylethylamine (DIEA) in anhydrous
DMF resulted in covalent attachment of the metal complexes to the DNA. Cleavage from
Olmon et al.
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