Charge Photoinjection in Intercalated and Covalently Bound
[Re(CO)
3
(dppz)(py)]
+
-DNA Constructs Monitored by Time
Resolved Visible and Infrared Spectroscopy
Eric D. Olmon
†
,
Pamela A. Sontz
†
,
Ana María Blanco-Rodríguez
‡
,
Michael Towrie
§
,
Ian P.
Clark
§
,
Antonín Vl
č
ek Jr.
‡,*
, and
Jacqueline K. Barton
*,†
†
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena,
California 91125, USA
‡
Queen Mary University of London, School of Biological and Chemical Sciences, Mile End Road,
London E1 4NS, United Kingdom
§
Central Laser Facility, STFC Rutherford Appleton Laboratory, Chilton, Didcot, Oxfordshire OX11
0QX, United Kingdom
Abstract
The complex [Re(CO)
3
(dppz)(py
′
-OR)]
+
(dppz = dipyrido[3,2-
a
:2
′
,3
′
-
c
]phenazine; py
′
-OR = 4-
functionalized pyridine) offers IR sensitivity and can oxidize DNA directly from the excited state,
making it a promising probe for the study of DNA-mediated charge transport (CT). The behavior
of several covalent and noncovalent Re-DNA constructs was monitored by time-resolved IR
(TRIR) and UV/visible spectroscopies, as well as biochemical methods, confirming the long-range
oxidation of DNA by the excited complex. Optical excitation of the complex leads to population
of MLCT and at least two distinct intraligand states. Experimental observations that are consistent
with charge injection from these excited states include similarity between long-time TRIR spectra
and the reduced state spectrum observed by spectroelectrochemistry, the appearance of a guanine
radical signal in TRIR spectra, and the eventual formation of permanent guanine oxidation
products. The majority of reactivity occurs on the ultrafast timescale, although processes
dependent on slower conformational motions of DNA, such as the accumulation of oxidative
damage at guanine, are also observed. The ability to measure events on such disparate timescales,
its superior selectivity in comparison to other spectroscopic techniques, and the ability to
simultaneously monitor carbonyl ligand and DNA IR absorption bands makes TRIR a valuable
tool for the study of CT in DNA.
INTRODUCTION
The ability of DNA to mediate charge transport (CT) has been established using a variety of
redox-active probes and in a great diversity of experimental systems.
1–3
The efficiency of
DNA-mediated CT is affected by several factors, including the extent of electronic coupling
between the probe and the DNA base stack, coupling within the base stack itself, the driving
force of the CT reaction, and the base sequence. DNA CT has been observed over long
molecular distances with little attenuation,
4–6
suggesting its utility in molecular-scale
*
To whom correspondence should be addressed. jkbarton@caltech.edu, a.vlcek@qmul.ac.uk.
Supporting Information. Strand sequences and MALDI data; Steady-state emission spectra of AT-30 and AT-30 with Re
′
-OEt before
correction for emission from DNA; histograms from maximum entropy analysis of ps time-resolved emission data; ps TRIR
difference spectra of Re-25(G) and Re-25(I) samples; detailed view of ns TRIR difference spectra in the DNA spectral region for the
GC-30 + Re
′
-OH sample. This material is available free of charge via the internet at http://pubs.acs.org.
NIH Public Access
Author Manuscript
J Am Chem Soc
. Author manuscript; available in PMC 2012 August 31.
Published in final edited form as:
J Am Chem Soc
. 2011 August 31; 133(34): 13718–13730. doi:10.1021/ja205568r.
NIH-PA Author Manuscript
NIH-PA Author Manuscript
NIH-PA Author Manuscript
devices
7–9
and in biological systems.
2,10–13
Many of the properties of DNA CT have been
elucidated in experiments involving the slow accumulation of oxidative damage at low
potential guanine sites. While such methods remain useful in the investigation of DNA CT,
a general probe for direct, time-resolved monitoring of these processes remains elusive.
Time-resolved infrared (TRIR) spectroscopy offers several advantages over other time-
resolved methods for the study of CT events.
14
With the proper choice of IR-active probe
and solvent medium, changes in the absorption pattern of well-resolved, transient IR bands
provide kinetic information on specific photophysical, chemical, and biochemical processes,
together with structural characterization of the excited states and reaction intermediates
involved. One common family of probes are coordination complexes of the type
[Re(CO)
3
(N,N)(L)]
n
, where N,N stands for an
α
-diimine ligand such as 2,2
′
-bipyridine
(bpy), phenanthroline (phen), or dppz (dipyrido[3,2-
a
:2
′
,3
′
-
c
]phenazine) and L represents an
axial ligand, often Cl (
n
= 0) or functionalized pyridine (
n
= 1+).
15–23
Photophysical or
photochemical reactions involving these Re complexes are manifested in TRIR spectra as
changes in the intensities and positions (energies) of absorption bands due to CO stretching
vibrations of the Re(CO)
3
group,
ν
(C
≡
O). Variation of the N,N and L ligands affords fine
control over the excited-state characters and energetics.
16,18–20,22–27
These complexes have
also proven useful as biochemical probes for fluorescence imaging,
28
for monitoring the
dynamics of structural fluctuations,
29,30
and especially, for triggering photoinduced electron
transfer (ET).
31
Information on ET kinetics and intermediates provided by TRIR is more
direct than that obtained using UV/visible time-resolved spectroscopic methods due to their
low specificity. Recently, the presence of tryptophan along the ET pathway in Re(CO)
3
(4,7-
dimethyl-1,10-phenanthroline)-modified azurin was shown to increase the rate of ET.
32–34
Although other coordination complexes, such as dicarbonyl Ru species, W(CO)
5
(4-
cyanopyridine), and [Ru(bpy)(CN)
4
]
2
−
have been employed as TRIR probes, tricarbonyl Re
complexes have been studied much more extensively.
14,16,35
Most studies have focused on
using TRIR to monitor protein ET. TRIR can also be used to monitor changes in the
vibrational frequencies and IR band intensities of organic functionalities in ET assemblies.
36
Of particular interest, TRIR spectra were recorded following the 267 nm excitation of the
four canonical nucleotides and of poly(dG-dC)·poly(dG-dC) and poly(dA-dT)·poly(dA-
dT).
37
In that work, the lifetimes of the transient states of the free nucleotides ranged from
2.2 to 4.7 ps, while those of the polymers were an order of magnitude longer. Upon 200 nm
photoionization of 5
′
-dGMP and poly(dG-dC)·poly(dG-dC), evidence for the formation of
the guanine radical was observed by TRIR as the growth of a transient band at 1702 cm
−
1.38
In other experiments, TRIR was used to observe the triplet state of thymine and of 2
′
-dT,
39
as well as to unravel the pH-dependent photophysics of 5
′
-G, 5
′
-GMP, and poly(G).
40
Importantly, these studies indicate that TRIR can be used to monitor photoinduced changes
of DNA and of [Re(CO)
3
(N,N)(L)]
n
simultaneously, making it possible to investigate both
the donor and the acceptor sites of Re-DNA CT assemblies. Although interactions between
Re complexes and DNA have been studied by UV/visible spectroscopy,
41,42
these
interactions had not been investigated by vibrational methods until very recently.
43,44
Here, TRIR spectroscopy is used in conjunction with other methods to observe the DNA-
mediated oxidation of guanine in DNA by photoexcited [Re(CO)
3
(dppz)(py
′
-OR)]
+
, where
py
′
-OR represents pyridine functionalized at the 4 position (Figure 1). The influence of
guanine on the photochemical behavior of the Re complex bound to DNA is investigated by
comparing results obtained in four different DNA contexts, including two in which the
complex is covalently tethered to specific locations on the duplex. The data presented show
that the photoexcited Re complex can oxidize guanine at a distance of several bases away by
DNA-mediated CT and that this process can be monitored on the ps to
μ
s timescale by
TRIR. The results of this study, in which TRIR is used for the first time to observe DNA-
mediated CT between photooxidants and guanine in well-defined covalent constructs, shows
Olmon et al.
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that the DNA sequence surrounding the metal complex binding site has a large influence on
the photophysics and photochemistry of the system.
EXPERIMENTAL SECTION
Materials
Most reagents for metal complex synthesis and coupling were purchased from Sigma-
Aldrich unless otherwise indicated. 3-(pyridin-4-yl)propanoic acid (py
′
-OH) was purchased
from Chess GmbH (Mannheim, Germany). Reagents for DNA synthesis were purchased
from Glen Research (Sterling, VA). All reagents were used as received.
Complex and Conjugate Synthesis
Preparation of [Re(CO)
3
(dppz)(py
′
-OH)](PF
6
) was adapted from previously described
methods.
41
Following the synthesis, the PF
6
−
counter anion was exchanged (QAE Sephadex
A-25 resin, GE Healthcare) for chloride ion in order to increase the solubility of the complex
in aqueous media. Because facile proton loss from the carboxylic acid-modified pyridine
ligand results in an overall neutral zwitterionic species, altering the extent of electrostatic
repulsion between complex molecules and of electrostatic attraction to DNA, the protected
ethyl ester version of the complex, [Re(CO)
3
(dppz)(py
′
-OEt)]
+
(py
′
-OEt = ethyl 3-
(pyridin-4-yl)propanoate), was used for some experiments.
Oligonucleotide Synthesis and Modification
Oligonucleotides were synthesized using standard solid-phase phosphoramidite chemistry
on an Applied Biosystems 3400 DNA synthesizer. Covalent tethers were appended to the 5
′
-
OH termini of resin-bound oligonucleotides as described by Holmlin.
45
The alkyl tether was
added to the DNA strand by successive treatment with carbonyldiimidazole and
diaminononane. Agitation of the resin-bound, amine modified DNA strands in the presence
of excess (5 mg) [Re(CO)
3
(dppz)(py
′
-OH)]Cl,
O
-(benzotriazol-1-yl)-
N,N,N
′
,N
′
-
tetramethyluronium hexafluorophosphate (HBTU), 1-hydroxybenzotriazole hydrate
(HOBT), and diisopropylethylamine (DIEA) in anhydrous DMF for 24 hrs resulted in
covalent attachment of the metal complex to the DNA. Cleavage from the resin was effected
by incubation in NH
4
OH at 60 °C for 6 h. Oligonucleotides were purified by reversed-phase
HPLC and characterized by MALDI-TOF mass spectrometry (see Supporting Information).
Oligonucleotide concentrations were determined by UV/visible spectrophotometry
(Beckman DU 7400). Annealing was accomplished by incubating solutions containing
equimolar amounts of complementary strands in buffer (10 mM NaP
i
, 50 mM NaCl buffer;
pH 7.0) at 90 °C for 5 min followed by slow cooling over 90 min to ambient temperature.
Assay for Oxidative DNA Damage
Oxidative DNA cleavage experiments were performed using a protocol adapted from
Zeglis
46
with the following adjustments. Oligonucleotides were labeled at the 3
′
-end by
incubating a mixture of 2
μ
L single-stranded DNA (4
μ
M), 5
μ
L [
α
-
32
P]-dTTP (Perkin
Elmer), 2
μ
L terminal transferase (TdT; New England Biolabs), 5
μ
L CoCl
2
solution
(included with TdT), and 5
μ
L terminal transferase reaction buffer (included with TdT) for 2
h at 37 °C. Before gel purification, strands were incubated at 90 °C for 20 min in 100
μ
L
10% aqueous piperidine to induce cleavage of damaged strands. Following purification and
annealing, samples (10
μ
L, 2
μ
M) were irradiated in parallel for 2 hrs using a solar simulator
(Oriel Instruments) fitted with a 340 nm internal long pass filter. Samples were then treated
with 0.2 units calf thymus DNA and 10% piperidine (v/v), heated for 30 min at 90 °C, and
dried
in vacuo
. After gel electrophoresis, oxidative damage was quantified by
phosphorimagery (ImageQuant). Sample counts are reported as % of total counts per lane
and were corrected by subtracting the dark control.
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Spectroelectrochemistry
UV/visible spectroelectrochemistry was carried out using a custom-built, optically
transparent, thin-layer electrode (OTTLE) cell (path length = 0.1 mm) consisting of vapor-
deposited platinum working and pseudoreference electrodes and a Pt-wire auxiliary
electrode.
47
The potential of the cell was controlled by a potentiostat (CH Instruments
Model 650A electrochemical workstation). Samples consisted of saturated solutions of metal
complexes in dry acetonitrile with 0.1 M Bu
4
NPF
6
electrolyte. Samples were degassed by
bubbling argon and introduced into the optical cell using a gas-tight syringe prior to
measurement. The cell was held at a reducing potential, and spectra were acquired on a
Hewlett Packard 8452A diode array spectrophotometer every 4 s until the sample was fully
reduced.
UV/Visible Emission and Transient Absorption Spectroscopy
Steady-state emission spectra were recorded on a Fluorolog-3 spectrofluorometer (Jobin
Yvon) using 2 mm slits. Scattered excitation light was rejected from the detector by
appropriate filters. Reported spectra are averages of at least five consecutive measurements.
All time-resolved UV/visible spectroscopic measurements were carried out at the Beckman
Institute Laser Resource Center. Nanosecond luminescence decay measurements and
transient absorption (TA) measurements were performed using the third harmonic (355 nm)
of a 10 Hz, Q-switched Nd:YAG laser (Spectra-Physics Quanta-Ray PRO-Series) as the
excitation source (8 ns pulse width, 5 mJ/pulse). Probe light was provided by a
synchronized, pulsed 75 W Hg-Xe arc lamp (PTI model A 1010), and detection was
accomplished using a photomultiplier tube (Hamamatsu R928) following wavelength
selection by a double monochromator (Instruments SA DH-10). Scattered light was rejected
using suitable filters. The samples were held in 1 cm path length quartz cuvettes (Starna)
equipped with stir bars. TA measurements were made with and without excitation, and were
corrected for background light, scattering, and fluorescence.
Picosecond emission decay measurements
48–51
were performed using the third harmonic of
a regeneratively amplified mode-locked Nd:YAG laser (355 nm, 1 ps pulse width after
amplification) as the excitation source and a picosecond streak camera (Hamamatsu C5680,
photon-counting mode) as the detector. Emission was observed under magic angle
conditions using a 550 nm long-pass cutoff filter.
TRIR Spectroscopy
The ULTRA instrument at the STFC Rutherford Appleton Laboratory has been used. It is
described in detail elsewhere.
52
In brief, a titanium sapphire laser-based regenerative
amplifier (Thales) produces 800 nm, ~50 fs pulses at a 10 kHz repetition rate. The laser
output is split in two parts, one of which is either frequency doubled or used to drive an
OPA (Light Conversion, TOPAS) equipped with SHG and SFG units to produce a pump
beam at 400 or 355 nm, respectively. The second pumps a TOPAS OPA, yielding signal and
idler beams that are difference frequency mixed to generate ~400 cm
−
1
broad mid IR probe
pulses. An optical delay line is used to introduce a delay between the pump and probe
beams, and the mid IR probe spectrum is recorded at a given time delay using two 128
element HgCdTe detectors (Infrared Associates). For ns-
μ
s measurements, the sample was
pumped with 355 nm, 0.7 ns FWHM pulses (AOT, AOT-YVO-20QSP/MOPO), and probed
with electronically synchronized 50 fs IR pulses.
53
The sample solutions were placed in a
round dip
ca
. 0.75 mm deep, drilled into a CaF
2
plate and tightly covered with a polished
CaF
2
window. The cell was scanned-rastered across the area of the dip in two dimensions to
prevent laser heating and decomposition of the sample. FTIR spectra measured before and
after the experiment demonstrated sample stability.
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Fitting Methods
TRIR data were simulated at each time delay as a series of Gaussian terms in order to extract
kinetic data from overlapping transient bands. The area of each Gaussian was calculated,
and kinetic decays were constructed as the change in area with delay time. Nanosecond
time-resolved emission, TRIR, and TA data were fit by nonlinear least-squares analysis
using IGOR Pro software (Wavemetrics). Model functions consisted of a linear series of
exponential terms of the form
y
(
t
) =
Σ
a
i
exp[
−
t
/
τ
i
], where
a
i
and
τ
i
are the pre-exponential
factor and lifetime, respectively, of the
i
th term. Up to three exponential terms were included
until reasonable fits were obtained. For time-resolved emission data, the percent relative
contribution reported in Table 1 represents the number of photons emitted at the probe
wavelength by each emissive population, and is calculated as
a
n
τ
n
/
Σ
a
i
τ
i
(the area under the
decay for the
n
th exponential term normalized to the total area under the decay curve). For
TRIR and TA data, the percent relative contribution represents the change in absorbance of
species
n
extrapolated to time
t
= 0, and is calculated as
a
n
/
Σ
a
i
.
Picosecond emission data were collected at 1 ns, 5 ns, and 50 ns time ranges and spliced
together before fitting. Data were compressed logarithmically in time prior to fitting in order
to decrease the bias of long time data on the fit. These data could not be fit well to a series of
exponential terms and were instead analyzed by the maximum entropy method using a
MATLAB (MathWorks) routine written at Caltech.
48–51
RESULTS
Research Strategy and Design of Re-DNA CT Assemblies
With the aim to establish DNA oxidation by electronically excited rhenium tricarbonyl-
diimine complexes, we have employed a newly developed Re sensitizer, [Re(CO)
3
(dppz)(py
′
-OR)]
+
(R = H, Re
′
-OH; or R = CH
2
CH
3
, Re
′
-OEt), which can be covalently linked to DNA
(Figure 1). Three design elements make this a promising probe for the study of DNA-
mediated CT. The first is the incorporation of TRIR-active carbonyl ligands. Re carbonyl-
diimine complexes are useful probes in TRIR spectroscopic experiments due to the intense
and well-resolved bands corresponding to carbonyl stretching modes. These modes are
extremely sensitive to changes in electron density distribution, molecular structure and
environment.
17,24,30,32,54,55
The second design element is the inclusion of the planar dppz
ligand. By incorporating dppz on the metal center, we ensure effective electronic coupling
with the DNA base stack. Indeed, the binding constants for intercalating dppz complexes
such as [Ru(bpy)
2
(dppz)]
2+
and [Ru(phen)
2
(dppz)]
2+
are greater than 10
6
M
−
1.56
While the
binding of complexes like [Re(CO)
3
(dppz)(py
′
-OR)]
+
is weaker (10
5
M
−
1
)
41,42,57
due to its
lower electrostatic charge, the decrease of the molar absorptivity of its near-UV absorption
band (
i.e
., hypochromicity) upon incubation with DNA, as well as an increase in the melting
temperature of the bound DNA duplex by approximately 5 °C (depending on the sequence),
indicate that this Re complex indeed binds by intercalation. The third design element is the
ability to covalently attach the complex to DNA via carboxyalkyl-modified pyridine
incorporated at the axial coordination site. The covalent link between the complex and the
DNA strand, while flexible, restricts diffusion of the unbound complex, ensuring a higher
percentage bound than if the complex were allowed to diffuse freely. In addition, the
covalent link enables us to define the DNA sequence at the binding region, eliminating
sequence effects as a variable. Physical models suggest that in the equilibrium geometry
(disregarding frayed end effects) tethering restricts binding to the region within three base
pairs from the end of the duplex.
The DNA duplexes used were designed to test for the effect of the DNA sequence on the
efficiency of DNA oxidation. For systems in which guanine, an effective hole trap, is placed
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