Ruthenium-containing P450 inhibitors for dual enzyme inhibition and DNA damage

Article abstract of DOI:10.1039/C6DT04405K

Cytochrome P450s are key players in drug metabolism, and overexpression in tumors is associated with significant resistance to many medicinal agents. Consequently, inhibition of P450s could serve as a strategy to restore drug efficacy. However, the widespread expression of P450s throughout the human body and the critical roles they play in various biosynthetic pathways motivates the development of P450 inhibitors capable of controlled local administration. Ruthenium complexes containing P450 inhibitors as ligands were synthesized in order to develop pro-drugs that can be triggered to release the inhibitors in a spatially and temporally controlled fashion. Upon light activation the compounds release ligands that directly bind and inhibit P450 enzymes, while the ruthenium center is able to directly damage DNA.

Full text of DOI:10.1039/C6DT04405K

View Article Online
View Journal
Dalton
Transactions
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: A. M. Zamoria, C.
A. Denning, D. K. Heidary, E. Wachter, L. A. Nease, J. Ruiz and E. Glazer, Dalton Trans., 2017, DOI:
10.1039/C6DT04405K.
This is an Accepted Manuscript, which has been through the
Volume 45 Number
1 7 January 2016 Pages 1–398
Royal Society of Chemistry peer review process and has been
accepted for publication.
Dalton
Transactions
Accepted Manuscripts are published online shortly after
An international journal of inorganic chemistry
www.rsc.org/dalton
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
author guidelines.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the ethical guidelines, outlined
in our author and reviewer resource centre, still apply. In no
event shall the Royal Society of Chemistry be held responsible
for any errors or omissions in this Accepted Manuscript or any
consequences arising from the use of any information it contains.
ISSN 1477-9226
PAPER
Joseph T. Hupp, Omar K. Farha et al.
Efficient extraction of sulfate from water using
framework
a Zr-metal–organic
rsc.li/dalton

Please do not adjust margins
Dalton Transactions
Page 1 of 9
View Article Online
DOI: 10.1039/C6DT04405K
Dalton Transactions
ARTICLE
Ruthenium-containing P450 inhibitors for dual enzyme inhibition
and DNA damage
Ana Zamora,[†]b Catherine A. Denning,[†]a David K. Heidary,a Erin Wachter,a Leona A. Nease,a José
Ruiz,b and Edith C. Glazer*a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Cytochrome P450s are key players in drug metabolism, and overexpression in tumors is associated with significant
resistance to many medicinal agents. Consequently, inhibition of P450s could serve as a strategy to restore drug efficacy.
However, the widespread expression of P450s throughout the human body and the critical roles they play in various
biosynthetic pathways motivates the development of P450 inhibitors capable of controlled local administration.
Ruthenium complexes containing P450 inhibitors as ligands were synthesized in order to develop pro-drugs that can be
triggered to release the inhibitors in a spatially and temporally controlled fashion. Upon light activation the compounds
release ligands that directly bind and inhibit P450 enzymes, while the ruthenium center is able to directly damage DNA.
www.rsc.org/
Introduction
Cytochrome P450s are essential enzymes that catalyze
challenging organic transformations for the biosynthesis and
metabolism of various key molecules, including steroids,
retinoic acid, and vitamin D. In addition, hepatic P450s are
responsible for the degradation of xenobiotics, and thus, play a
central role in drug metabolism and deactivation. One
problematic issue is that their substrates include many
anticancer agents, decreasing the effective drug concentration
in the body. Compounding this problem, some P450s are
found specifically in tumors, where they are over expressed
and play a direct role in cancer initiation, progression, and
drug resistance. For example, CYP1B1 has been shown to
metabolize procarcinogens to carcinogens to initiate DNA
damage, and subsequently induce resistance to DNA damaging
chemotherapeutics.1-3 Alternatively, CYP19A (aromatase)
converts androgens to estrogens, and is an important target in
the treatment of estrogen driven cancers.4-8 CYP17A1 is
responsible for androgen synthesis, and abiraterone is a first-
in-class steroidal inhibitor of this enzyme used in late-stage
prostate cancer.9-12
Scheme 1. Design of dual action inhibitors.
To date, P450 inhibitors have been used as treatments for
the inhibition of steroid biosynthesis, as for breast and
prostate cancer, and for other indications, such as Cushing’s
disease.13-15 The dangers associated with clinical use of P450
inhibitors is that they are generally not isoform selective (<103
difference in Kd), and long-term systemic inhibition of P450s
can result in adverse drug interactions and altered hormone
levels.16,
17
An alternative approach to avoid these
consequences would be to develop agents that can be
activated to selectively inhibit desired P450 enzymes in a
spatially and temporally regulated manner. In the context of
anti-cancer agents, inhibition of P450s in cancerous tissues is
also a rational strategy to sensitize the cells to DNA damaging
agents,1 while reducing the bioactivation of procarcinogens as
a cancer driver. Furthermore, if the inhibition of the P450
could occur concurrently with the local administration of a
cytotoxic agent only within the tumor, deactivation of the drug
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
Please do not adjust margins

Please do not adjust margins
Dalton Transactions
Page 2 of 9
View Article Online
DOI: 10.1039/C6DT04405K
ARTICLE
Journal Name
by hepatic P450s could be avoided. Accordingly, we have bipyridine) or the corresponding bis-aqua Ru(bpy)2(OH2)2 in
synthesized dual action Ru(II) complexes as pro-drugs that can EtOH/H2O (1:1) while protected from light (see the
be triggered with light to simultaneously release a P450 Experimental section). Abstraction of the chlorides was carried
inhibitor and a DNA damaging metal center.
out with a silver salt in order to diminish the percentage of the
undesired monocoordinate complexes, [Ru(bpy)2LCl]+, which
complicated the purification. All complexes were formed in
good yields (38–73% yield) and exhibited moderately intense
metal-to-ligand charge transfer (MLCT) bands centered around
Photocaging is a well-established means to selectively release
biologically active agents with temporal and spatial control.18 Metal
complexes have been used as photocaging groups with great
success, with pioneering work by Etchenique,19-21 Franz,22-24 and
Kodanko.25-27 Our approach differed slightly from traditional
photocaging, however, as the metal complex is intended to act as a
caging group that would transform into an active biological effector
in its own right, in addition to the ligand that it protected in the
intact complex form.
450–490 nm. Not surprisingly, compound
2 was isolated as a
mixture of the possible coordination isomers, as either pyridyl
ring in the asymmetric ligand (the A- or B-ring, as shown in
Chart 1) can coordinate to the Ru(II) center.
Light-triggered release of the coordinated ligands was
monitored under different solvent conditions by UV/Vis
absorption spectroscopy, HPLC, and mass spectrometry. All
complexes were able to cleanly release both monodentate
ligands in acetonitrile (MeCN) after irradiation with blue light
(470 nm). A biphasic blue-shift was observed in their spectra
(Figures S6-S8), and a common final 425 nm band was found
In order to test this strategy, three P450 inhibitors were
chosen that could be coordinated to Ru(II) complexes. These
compounds all contain nitrogen heterocycles, and thus are able to
directly ligate both the Ru(II) center and, after photorelease, the
iron heme in P450s. Metyrapone and etomidate have been
primarily used to inhibit P450 11B1, also known as steroid 11-beta-
monooxygenase.28 Both compounds appear to bind similarly in the
binding site of CYP11B1, where the N-heterocycle is capable of
ligating the catalytic heme iron while the other ring interacts with
Arg110 and Phe130 via π-stacking.29 A third, novel small molecule,
compound 1, was synthesized;30 it has the metyrapone molecular
skeleton with etomidate features: imidazole and benzene rings
(Chart 1).
for 2–4, which indicated the formation of a unique product,
[Ru(bpy)2(MeCN)2]2+, after the release of the corresponding
inhibitors.
The photoproducts formed in aqueous solution were
identified using HPLC and mass spectrometry analysis (Figures
S9-S14). The chromatograms showed the appearance of new
signals in irradiated samples, which corresponded to the free
inhibitor and the mono-aqua Ru(II) complex. This was
confirmed by the mass spectrum of the solution, with peaks
corresponding to [Ru(bpy)2L(H2O)]2+, [Ru(bpy)2L]2 and [L+H]+
(Figures S10, S12, S14). It is to be noted that the UV/Vis profile
Chart 1. Structures of P450 inhibitors and Ru(II) complexes.
of the products for 3 and 4 did not differ significantly from that
of the complex protected from light. The extent of the
photoejection reaction varied from 40 to 65%. This
observation is consistent with other [Ru(bpy)2L2]2+ and
[Ru(phen)2L2]2+ complexes, where substitution of the second
N-monodentate ligand, L, requires much longer irradiation
times or does not occur.19,25,
31-34
Finally, in the dark, all
complexes were stable at room temperature and at 37 °C
when measured over 48 hrs (Figure S5).
In order to directly investigate the interactions of the metal
complexes with a P450, cytochrome P450BM3 (CYP102A1), a
soluble bacterial P450, was chosen as a model system. A
mutant form of P450BM3 has been shown to recapitulate the
activity of mammalian drug metabolizing P450s,35 which has
made P450BM3 a commonly used experimental system.36
Results and discussion
Ruthenium complexes
2–4 (Chart 1) were synthesized by
refluxing the respective inhibitors with Ru(bpy)2Cl2 (bpy = 2,2’-
A
C
B
0.2
0.1
0.0
0.2
0.2
0.1
0.0
P450
P450 + inhibitor
P450 + Ru(II) dark
P450 + Ru(II) light
0.1
0.0
300
400
500
600
700
300
400
500
600
700
300
400
500
600
700
Wavelength (nm)
Figure 1. Absorption spectra of P450BM3 inhibitor saturated and Ru(II) dark and light systems: 2 (A), 3 (B), 4 (C). The ratio used was P450: Complex (1:10) and P450:
Wavelength (nm)
Wavelength (nm)
Ligand (1:4) for each of the respective ligands used to generate the complexes.
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
Dalton Transactions
Page 3 of 9
View Article Online
DOI: 10.1039/C6DT04405K
Journal Name
ARTICLE
Importantly, while mammalian P450s are generally membrane
associated, which complicates analysis, P450BM3 is soluble and
thus amenable to various spectroscopic investigations as well
as enzyme turnover assays. Furthermore, the in vitro system
was chosen due to the intrinsic complications of studying P450
inhibition in cells or cell lysates, due to the need to create cell
lines that overexpress the enzyme and reductase partners, and
to provide exogenous reductants such as NADPH.
Figure 3. Agarose gels showing the dose response of 2 (A), 3 (B), and 4 (C) with 40
μg/mL pUC19 plasmid with and without irradiation (λ>470 nm). Dark, left; 1 hr
irradiation, middle; 3 hr irradiation, right. Lane 1 and 12: DNA ladder; Lane 2: EcoRI;
Lane 3: Cu(OP)2; Lanes 4–11: 0, 7.8, 15.6, 31.3, 62.5, 125, 250, 500 μM (500 μM
corresponds to a metal center:base ratio of 4:1). EcoRI and Cu(OP)2 are used as
controls for linear and relaxed circular DNA. EtBr was used to visualize the DNA.
As all three free P450 inhibitors directly ligate the heme
iron, either through a pyridyl or imidazole ring, a type II
spectra shift was observed upon inhibitor binding. A 7 nm red-
shift in the heme soret was observed for metyrapone,
etomidate, and compound 1, with the appearance of a second
minor peak around 360 nm, where a shoulder is observed in
the free enzyme (Figure 1). Difference spectra for all
compounds are shown in Figure S17, with trough maxima
around 410 nm. Significant shoulders are observed on the
troughs at 390 nm, consistent with a mixture of type IIa and
type IIb spectra37-39 (type IIa spectra are observed when the
enzyme is in the high-spin state in the absence of the ligand;
type IIb is seen when the enzyme is in the low-spin state).37
exhibited inhibition of enzyme activity (Figure 2 and Figure S18). As
anticipated, the three Ru(II) complexes all demonstrated triggerable
enzyme inhibition. Compound 4 gave the largest window for dark
vs. light activity (Figure 2), with IC50 values for enzyme inhibition of
6.8 and 0.05 ꢁM, respectively. This compound thus provides a 136-
fold difference between activity in the dark and the light. The IC50
value for the free ligand 1 was 0.06 ꢁM, in excellent agreement
with the activity of the complex in the light. Each light activated
system displayed very similar activity to the free inhibitor (Figure
2B; compound 2 IC50 = 0.19 ꢁM vs. 0.75 ꢁM for metyrapone;
compound 3 IC50 = 0.04 ꢁM vs. 0.02 ꢁM for etomidate).
The ꢀAmax between the peak and trough and the intensity of
the shoulder varied as a function of the nitrogen containing
coordinating ligand, as previously reported.38
Upon exposure to an Indigo LED array (28 J/cm2), each
complex induced the same mixed type IIa and IIb spectral shift
observed with the free inhibitors. Conversely, when the Ru(II)
To extend the investigation to
a medically validated,
commonly used experimental system, pooled human liver
microsomes (HLMs) were used to study the light-triggered
inhibition of enzyme turnover. HLMs contain membrane-bound
human CYPs which play major roles in first pass metabolism of
xenobiotics.50, 51 Incubation of HLMs with compound allows study of
how these compounds impact the activity of multiple CYPs at once,
and in a membrane environment. This provides a good model for
human CYPs. Compound 4 exhibited the largest window for light-
mediated enzyme inhibition for P450BM3, so it was tested along with
the free ligand 1. In the dark, at 100 ꢁM, compound 4 was found to
minimally inhibit CYPs in HLMs, but complete inhibition was
observed after irradiation (Figure 2C). A similar trend was seen with
free ligand 1, with nearly complete inhibition of enzyme turnover.
Having confirmed the utility of the light active compounds for
inhibition of cytochrome P450 activity, DNA damage was
investigated using agarose gel electrophoresis. Dose responses
were performed with compound 2–4 with pUC19 plasmid, and the
solutions were either protected from light or irradiated for 1 or 3
hrs (60.4 or 181.4 J/cm2) before incubation at 37 °C for 12 hrs
(Figure 3). No effect was seen for any of the intact pro-drug forms
of the complexes, but all induced DNA damage upon light
activation. DNA damage was visualized by reduced mobility of DNA,
complexes
3 and 4 were kept in the dark there as little or no
observed change the P450BM3 absorption spectra, indicating
that the complexes function as pro-drugs and do not directly
affect the active site of the enzyme. Complex 2, containing the
pendant metyrapone, did exhibit a slight change in absorption
profile, suggesting the intact complex containing the pendant
pyridyl ligand was capable of interacting with the enzyme
enough to perturb the absorption spectra. While unexpected,
previous structural studies have shown that small molecules
that are tethered to large fluorophores40-42 or even metal
complexes43-47 can bind P450s and induce opening of the
substrate channel. Interestingly, the difference spectrum
(Figure S17A) indicates a different binding mode than either
free metyrapone or the light-activated 2, and appears to be
more a type I spectrum, consistent with displacement with
water from the heme, but with no direct ligation of a
coordinating nitrogen.37
Enzyme inhibition was tested using resorufin ethyl ether as a
substrate in a fluorescence based assay.48, 49 Each of the free ligands
C
A
B
10
8
6
4
2
12
100
80
60
40
20
0
8
4
0
0.2
0.1
0.0
0
5
10
15
20
25
30
-2
-1
0
1
Time (min)
Log concentration (ꢁM)
Figure 2. (A) Relative activity of P450BM3 in the presence of 1 (red diamonds) and 4 in the dark (black circles) and following irradiation (blue squares). (B) IC50 values for all three
complexes and the respective free ligands. (C) Inhibition of CYPs in HLMs by free ligand 1 (red circles) and complex 4 (blue squares) after irradiation compared to no
compound (black circles) and 4 in the dark (green triangles).
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
Dalton Transactions
Page 4 of 9
View Article Online
DOI: 10.1039/C6DT04405K
ARTICLE
Journal Name
B
A
cytochrome P450 inhibitor. The best system, compound
4,
exhibited a 136-fold difference in protein inhibition when
irritated with light as compared to in the dark, with an IC50 of
0.05
complexes did not damage DNA or interfere with its function,
as indicated by gel electrophoresis and activity in
ꢁM upon activation. In the absence of irradiation, the
a
transcription and translation assay. Upon light activation,
protein production was inhibited with an IC50 between 5 and
10 ꢁM in the light. Furthermore, the metal center damages the
DNA even in the presence of protein, indicating that DNA is the
Figure 4. (A) An in vitro transcription and translation experiment allows for
detection of DNA damage that results in inhibition of GFP production. Compound 4
was incubated in the presence of both plasmid and P450BM3 at equal
concentrations. (B) GFP production was unaffected by the addition of P450BM3 (+
P450; lane 2) and the protein had no impact on the ability of compound 4 to
damage DNA upon light activation (lanes 3–6).
preferred target.
The choice to inhibit P450 enzymes is a key feature in the
design of these pro-drugs. The targeting of enzymes implicated
in drug resistance could result in synergistic activity for DNA
damaging agents in cancer cells and tissues, though more
involved studies in cancer cell lines engineered and optimized
to detect P450 activity and inhibition will be required for full
validation of this potential therapeutic approach. However, the
clear inhibition of P450 activity and the DNA damage and
suppression of transcription and translation in vitro, combined
with the well-established cytotoxicity of light activated, ligand
deficient Ru(II) complexes in cells are strongly promising. This
is also, to the best of our knowledge, the first report of
consistent with direct adduct formation, as demonstrated in
classical experiments on cisplatin52-54 and more recently with
56
analogous light-activated ruthenium compounds.55,
Very little
relaxed circular DNA was observed, but what was formed likely
resulted from single strand breaks induced by singlet oxygen (1O2).
Ligand ejection proceeds from an excited state that is populated
from the 3MLCT state, which also generates 1O2; thus, there is a
competition in relaxation pathways, and complexes that have
longer t1/2 values have been found to induce more single strand photocaged P450 inhibitors. These compounds may be useful
58
breaks. We57,
and others59 have thus observed that the
for basic research applications as tools that provide spatial and
temporal control over P450 inhibition, and could answer
several open questions in the role that P450s play in malignant
cell transformation and drug resistance. Single mode of action
photocaged systems, which do not damage DNA, are also
under development to allow for the triggered control of P450
activity without complications from the activity of the metal
center.
combination of the different relaxation pathways makes these
agents capable of “dual photoreactivity” independent of the
biological activity of the released ligands. However, little 1O2 was
detected, as shown in Figure S19, consistent with the DNA damage
study and relaxation primarily through ligand ejection. Finally, all of
the free ligands were tested as controls, and as anticipated, they
had no impact on the plasmid DNA (Figure S20).
The ultimate experiment was to investigate if the light
activated metal complexes were able to inhibit DNA function
in the presence of the P450 enzyme. Accordingly, an in vitro
60
transcription and translation experiment was performed, as
Experimental Section
shown in Figure 4A. A plasmid coding for green fluorescent
protein (GFP) was incubated with compound
4 in the dark or
following activation with light. The experiment was also Materials and instrumentation
performed in the presence of P450BM3. No impact on GFP
Chemicals used for synthesis were purchased from VWR or
Fisher Scientific and used without further purification. cis-
Dichlorobis(2,2’-bipyridine)ruthenium(II) dihydrate was purchased
from Strem chemicals. Human liver microsomes were purchased
from Sekisui Xenotech. The Singlet Oxygen Sensor Green was from
Molecular Probes.
A Varian Mercury spectrometer was used to obtain 1H NMR
(400 MHz) and 13C (100 MHz) spectra. Chemical shifts are reported
relative to the solvent peak (CD3CN – δ 1.94 or CDCl3 – δ 7.24 for 1H
NMR; CD3CN – δ 1.39 for 13C NMR). Electrospray ionization (ESI)
production was observed for
4 in the dark, while a dose
dependent inhibition of protein production was found
following activation with light, as shown in Figure 4B. The
addition of P450BM3 had no impact on GFP transcription or
translation in the absence of the Ru(II) complex, or in its
presence. Thus, the P450BM3 enzyme does not serve as a “sink”
for the activated metal center, and does not interfere with the
DNA damage mechanism. This validates that the Ru(II) center
can target DNA while the released ligands target the P450
enzyme.
mass spectra were obtained using
a Varian 1200L mass
spectrometer at the University of Kentucky Environmental Research
Training Laboratory (ERTL). Absorption spectra for the extinction
coefficient determination, acetonitrile photoejection and binding
constant (Kd) determination for ligands were obtained on a Cary 60
UV/Vis spectrophotometer. Full spectrum absorbance readings to
study the photoejection for complexes 1–3 in different aqueous
media were obtained using a BMG Labtech FLUOstar Omerga
Conclusions
The three complexes described in this report serve as proof-of-
concept systems for single agent “drug cocktails” and
demonstrate that, upon light activation, the metal center is
able to damage DNA while the liberated ligand acts as a
4 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
Dalton Transactions
Page 5 of 9
View Article Online
DOI: 10.1039/C6DT04405K
Journal Name
ARTICLE
microplate reader. Binding saturation studies for each compound as MHz): δ 159.57, 159.48, 159.01, 158.96, 158.47, 158.37, 154.10,
well as Kd determination for irradiated complexes 1–3 were 154.00, 153.96, 153.83, 142.93, 142.82, 141.88, 141.52, 138.42,
completed using an Agilent 8453 UV/Vis spectrometer. Compound 138.35, 138.16, 138.10, 129.90, 129.23, 129.09, 128.46, 128.10,
purity was determine with an Agilent 1100 Series HPLC using a 128.03, 126.95, 126.63, 126.10, 126.05, 124.70, 124.48, 62.41,
previously reported method.61 Light activation for photoejection 57.86, 57.62, 22.35, 22.12, 14.46, 14.42 ppm; ESI MS C48H48N8O4Ru:
-
-
experiments was achieved using a 470 nm LED array (16.7 mW/cm2) m/z calcd [M]+ PF6 1047.25, [M]2+ 451.14, found 1047.1 [M]+ PF6 ,
from Elixa. An Indigo LED Flood Array (466 mW/cm2) from Loctite 451.1 [M]2+. Purity by HPLC: 98.3 % by area; UV/Vis in CH3CN, λmax (ε
was used for light activation for the enzyme assays. A Tecan
SPECTRAFluorPlus Plate Reader was used to determine change in
fluorescence for the enzyme activity assay and IVTT assay. Agarose
gels were digitally imaged using a BioRad ChemiDoc System.
M
-1 cm-1) = 235 (50900), 290 (53900), 325 (8900), 475 (8200).
[Ru(bpy)2(1)2](PF6)2 (4):
Silver nitrate (65.2 mg, 0.384 mmol) was added to a suspension
of [Ru(bpy)2Cl2]·2 H2O (100 mg, 0.192 mmol) in water (15 mL), and
the mixture was stirred overnight at RT. The solution was filtered
Compound synthesis, characterization, and ion exchange
2-(1-Imidazolyl)-2-methyl-1-phenyl-2−1-propanone) (1) was
under
N2.
2-(1-Imidazolyl)-2-methyl-1-phenyl-2−1-propanone;
synthesized following a previously published procedure.52 All metal compound 1) (102.8 mg, 0.480 mmol) and 15 mL of EtOH were
added to the solution, which was stirred at 85 °C under N2 for 24 hr.
After cooling the reaction, the solution was concentrated, 1–2 mL
of a saturated aqueous KPF6 solution was added and the precipitate
was extracted into CH2Cl2 (3x15 mL). The crude was purified by
column chromatography using H2O:MeCN:KNO3 as eluent (from
0:100:0 to 10:90:0.2). The product was obtained in 73% yield (158
complexes were synthesized under low ambient light and were
protected using aluminum foil throughout each step of synthesis,
isolation, and characterization. Silver salts were used to facilitate
ligand exchange; the choice of the specific salts in the different
reactions was due only to reagent availability.
mg) as a crystalline red solid. 1H NMR (CD3CN, 400 MHz): δ 8.66 (d,
= 5.2, 2H), 8.22 (d, = 8.0 Hz, 2H), 8.08 (d, = 8.0 Hz, 2H), 7.99 (t,
J
[Ru(bpy)2(Met)2](PF6)2 (2):
J
J
J
=
[Ru(bpy)2Cl2]·2 H2O (125 mg, 0.240 mmol) was dissolved in
water (7 mL) under N2 at 80 °C. To this 2-methyl-1,2-di-3-pyridil-1-
propanone (136 mg, 0.6 mmol) was added, and the red solution
was stirred overnight at 80 °C. The resulting solution was cooled to
room temperature (RT) and extracted into CH2Cl2 (3x10 mL) to
remove the excess free ligand. The complex was precipitated out of
the aqueous phase with 1–2 mL of a saturated aqueous KPF6
solution and extracted with CH2Cl2/MeCN (3x10 mL). The crude
complex was purified by column chromatography using
H2O:MeCN:KNO3 as eluent (from 0:100:0 to 12:87.2:0.8). The
product was obtained in 38% yield (104 mg) as an orange solid. ESI
MS C48H44N8O2Ru: m/z calcd [M]2+ 433.13, found 433.2 [M]2+. Purity
by HPLC: 99.3 % by area; UV/Vis in CH3CN, λmax (ε M-1 cm-1) = 290
(43200), 345 (12100), 445 (8400). Note: NMR was not completed
due to the compound containing a mixture of isomers.
8.0 Hz, 2H), 7.75 (m, 4H), 7.53 (m, 4H), 7.29 (s, 2H), 7.25 (s, 2H),
7.17 (m, 6H), 7.06 (d,
= 7.6 Hz, 4H), 6.53 (s, 2H), 1.78 (s, 12H); 13C
J
NMR (CD3CN, 100 MHz): δ 158.67, 158.16, 153.58, 153.25, 139.57,
137.97, 137.54, 135.15, 134.23, 130.68, 129.61, 128.80, 128.12,
127.76, 124.54, 124.26, 120.71, 67.75, 30.99, 27.54, 27.26 ppm; ESI
-
MS C46H44N8O2Ru: m/z calcd [M]+ PF6 987.23, [M]2+ 421.13, found
-
987.4 [M]+ PF6 , 421.1 [M]2+. Purity by HPLC: 97.5 % by area; UV/Vis
in CH3CN, λmax (ε M-1 cm-1) = 245 (41200), 290 (50400), 335 (8200),
485 (8300).
-
Compounds 2–4 were converted to Cl salts by dissolving 5–20
mg of product in 1–2 mL methanol. The dissolved product was
loaded onto an Amberlite IRA-410 chloride ion exchange column,
eluted with methanol, and the solvent was removed in vacuo
.
The purity of each Ru(II) complex was analyzed using the
method in Table S1 (mobile phases of 0.1% formic acid in dH2O and
0.1% formic acid in HPLC grade CH3CN). Samples of each Ru(II)
complex were prepared in dH2O and protected from light before
injection on the HPLC.
[Ru(bpy)2(Eto)2](PF6)2 (3):
Silver triflate (99 mg, 0.384 mmol) was added to a suspension of
[Ru(bpy)2Cl2]·2 H2O (100 mg, 0.192 mmol) in water (15 mL) and the
mixture was stirred overnight at RT. The solution was filtered under
N2. Etomidate (94 mg, 0.384 mmol) and 15 mL of EtOH were added
to the solution, which was then stirred at 85 °C under N2 for 24 hr.
After cooling the reaction, the solution was concentrated, 1-2 mL of
a saturated aqueous KPF6 solution was added, and the precipitate
was extracted into CH2Cl2 (3x15 mL). The crude was purified by
column chromatography using H2O:MeCN:KNO3 as eluent (from
0:100:0 to 20:80:0.4). The product was obtained in 47% yield (108
Photoejection studies
MeCN photoejection studies:
-
Photoejection studies were performed on the PF6 salts of 2–4
(30 μM) in 3 mL of acetonitrile in a 1 cm pathlength quartz cuvette
placed 12 inches below a 470 nm LED array in duplicate. Each
sample was prepared from the dissolution of the pure solid in
acetonitrile and diluting it to the above final concentration. The
samples were protected from ambient light until irradiated with the
LED array. Ligand ejection was monitored by taking absorption
spectra after specific time points until the spectra ceased to evolve.
The half-life (t1/2) of photoejection was determined by plotting the
difference in absorbance between two points around the isosbestic
point versus time using Graphpad Prism software.
mg) as a crystalline red solid. 1H NMR (CD3CN, 400 MHz): δ 9.03 (d,
= 5.6 Hz, 1H), 8.97 (d, = 5.2 Hz, 1H), 8.35 (d, = 8.4 Hz, 2H), 8.27 (d,
= 8.4 Hz, 1H), 8.12 (q, = 8.4 Hz, 2H), 8.00
= 5.2 Hz, 1H), 7.87 (m, 3H), 7.70 (m, 3H), 7.49 (s, 1H), 7.37-7.19
= 6.4 Hz, 2H), 6.72 (d, = 7.2 Hz, 2H), 6.29 (q,
7.0 Hz, 1H), 6.19 (q, = 7.2 Hz, 1H), 4.15 (m, 4H), 1.75 (d, = 7.2 Hz,
3H), 1.71 (d,
= 7.2 Hz, 3H), 1.19 (m, 6H); 13C NMR (CD3CN, 100
J
J
J
J
= 8.0 Hz, 1H), 8.24 (d,
J
J
(d,
J
(m, 10H), 6.88 (d,
J
J
J =
J
J
J
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 5
Please do not adjust margins

Please do not adjust margins
Dalton Transactions
Page 6 of 9
View Article Online
DOI: 10.1039/C6DT04405K
ARTICLE
Journal Name
Aqueous photoejection studies:
Protein was further purified by loading onto a Hi-Prep 26/60
Photoejection studies using the Cl- salts of 2–4 in aqueous Sephacryl S200 HR (GE Healthcare) sizing column equilibrated with
media (water, 1X PBS and Opti-MEM with 1% FBS) were performed gel filtration buffer (20 mM Tris, 150 mM NaCl, pH 8.0).
in triplicate using a Greiner UV clear half-area 96-well plate. The
All fractions with a 420/280 nm ratio above 1.2 were
kinetics for ligand ejection were determined for 2–4 (40 µM) with a concentrated using Ultracel-30K Millipore centrifugal units. For
final volume of 200 µL. The well plate was positioned 12 inches storage, glycerol was added to give a final concentration of 50%,
below a 470 nm LED array, and full spectra were collected after set the protein was aliquoted, snap frozen, and stored at –80 °C.
Prior to using PM BM3, glycerol was removed and the buffer
was exchanged to assay buffer (20 mM Tris, 20 mM NaCl, 10 mM
CaCl2, pH 7.4) using a PD-10 desalting column (GE Healthcare). To
determine protein concentration, a CO binding assay was used as
previously described.62
time points of light exposure for a total of 5 hrs. The change in
absorbance was plotted using the same method as described for
acetonitrile.
Photoejection reactions were followed by HPLC using 40 µL
injections of 200 µM solution of each Ru(II) complex prior to
irradiation and after 1 min irradiation with 470 nm light. The free
ligands were used as controls. Complexes 2 and 4 were run using
the method described in Table S1, and complex 3 was run using the
method described in Table S2. Samples of each Ru(II) complex were
prepared in dH2O and protected from light before injection when
necessary. Ligands were fully dissolved in DMSO and diluted in
dH2O, so that the final solution contained 1% DMSO. Products were
characterized by the retention time (tR) as well as the UV/Vis profile
corresponding to the HPLC peak. The extension of the reaction was
calculated by integration of the area corresponding to the free and
photoejected ligand. Finally, the irradiated sample was also
analyzed by ESI MS.
P450BM3 binding affinity
To determine if the inhibitors could saturate PM BM3, the
protein was added to a 3 mL 1 cm pathlength quartz cuvette at a
final concentration of 2.5 µM. UV/Vis spectra were taken before
and after the addition of compound. Ligands were tested at 10 µM,
whereas Ru(II) complexes were tested at 25 µM in the dark and
after 1 min irradiation. After the addition of compound the samples
were allowed to incubate at RT for 30 sec before data collection.
Absorbance binding titrations of ligands and light activated
complexes 2–4 were performed in a 1 cm pathlength quartz cuvette
with 2.5 µM protein and a total volume of 3 mL. The absorbance
was measured after each ligand or Ru(II) addition from 0–64.0 µM
(metyrapone), 0–30.3 µM (etomidate and 1), 0–64.7 µM (2), 0–54.2
µM (3) and 0–45.8 µM (4). The Ru(II) only absorbance was
measured and blanked in parallel. Binding constants were
determined by plotting the change in absorbance at 425 nm vs.
concentration of ligand or Ru(II). Data was plotted using Graphpad
Prism software and fit using a one site-total binding equation.
Expression and purification of P450BM3
The pCWori vector containing the gene for the heme domain
(Thr 1–Thr 463) of P450BM3 with five mutations incorporated (R47L,
F81I, F87V, L188Q, E267V, “PM BM3”) was transformed into
BL21(DE3) cells. After transformation, cells were grown overnight
on 50 μg/ml carbenicillin plates at 37 °C.
Small 5 mL growths in Luria Broth (LB) with 100 μg/mL
ampicillin were grown overnight and then added to 1 L of Terrific
Broth (TB) with 100 μg/mL ampicillin and 0.4% glycerol. Cells were
grown at 180 rpm and 37 °C until an OD600 of 0.6–0.8 was reached.
Protein production was induced by addition of 0.5 mM isopropyl β-
D-1 thiogalactopyranoside (IPTG). Temperature and shaking were
decreased to 30 °C and 150 rpm, respectively. After 16-20 hrs, the
cells were harvested by centrifugation at 4,000 rpm for 15 min at 4
°C. The supernatant was decanted and the cell pellet was
resuspended in lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM
imidazole, 0.1 mM EDTA, and 0.1 μM phenylmethylsulfonyl fluoride
(PMSF), pH 8.0). The resuspended pellet was sonicated on ice using
a Branson Sonifer 250 microtip, for 15 min with output control of 3
and duty cycle of 50%. The lysate was then centrifuged for 1 hr at
17,000 x g and 4 °C.
P450BM3 inhibition assay
Inhibition assay with purified PM BM3:
An enzymatic turnover assay was utilized to determine the
inhibition of resorufin ethyl ether metabolism by PM BM3 in the
presence of added compound. Each compound was added to 250
nM PM BM3 in 1X PBS (phosphate buffered saline, pH 7.5) at
varying concentrations between 0–10 μM in Greiner clear 96 well
plates and incubated for 10 min. Ru(II) complexes were tested in
the presence and absence of light, where stock solutions were
irradiated for 1 min prior to incubation with PM BM3. Following
incubation with compound, 5 μM resorufin ethyl ether was added
and incubated at RT for 5 min. To initiate enzymatic turnover, 5 mM
hydrogen peroxide was added, and changes in fluorescence of
resorufin ethyl ether were monitored over 5 min using a Tecan
spectrafluor plus microplate reader at excitation 535 nm and
emission 595 nm.
The supernatant was decanted and syringe filtered with a 0.45
μm polytetraflurorethylene filter prior to addition to a His-Trap
column (GE Healthcare) equilibrated in buffer A (50 mM NaH2PO4,
300 mM NaCl, and 20 mM imidazole, pH 8.0). PM BM3 was eluted
using a linear gradient of 20 mM to 200 mM imidazole with buffer B
(50 mM NaH2PO4, 300 mM NaCl, and 200 mM imidazole, pH 8.0).
Fractions were collected based on color and absorbance at 420 nm
and 280 nm. PM BM3 containing fractions were then concentrated
using Ultracel-30K Millipore centrifugal units at 4500 x g and 4 °C.
Inhibition assay with Human liver microsomes (HLMs):
An enzymatic turnover assay was utilized to determine the
inhibition of resorufin ethyl ether metabolism by enzymes
responsible for first-pass metabolism in pooled human liver
microsomes (HLMs) in the presence of added compound. 125 μM of
1 or 4 was added to 20 mg/mL HLM to a final concentration of 100
μM in 100 mM KH2PO4, 10 mM MgCl2 buffer, pH 7.5. After
6 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
Dalton Transactions
Page 7 of 9
View Article Online
DOI: 10.1039/C6DT04405K
Journal Name
ARTICLE
incubation with compound for 10 min, 5 μM resorufin ethyl ether supported by Fundación Séneca-CARM (Exp. 19020/FPI/13).
was added followed by 1.3 mM NADPH to initiate enzymatic The Spanish Ministerio de Economía y Competitividad and
turnover. Changes in fluorescence of resorufin ethyl ether were FEDER (Project CTQ2015-64319-R) is also acknowledged. E. W.
monitored over 30 min using a Tecan spectrafluor plus microplate was supported by the University of Kentucky Research
reader at the same settings as above. Compound 4 was incubated Challenge Trust Fund Fellowship and C. A. D. by NIDA T32
with HLMs in the dark and after 1 min irradiation with the Indigio Research Fellowship (NIH DA016176).
LED.
In vitro transcription and translation
Notes and references
A 1-Step Human Coupled IVT Kit–DNA (Thermo Scientific) was
used to carry out the experiment.60 For each reaction, 0.5 µg of the
pCFE-GFP plasmid and 5–20 μM 4 was used. Complex 4 was either
irradiated for 1 min with the Loctite Indigo LED or kept in the dark.
Prior to carrying out the IVT reaction, 4 was incubated with the
plasmid overnight in the presence or absence of PM BM3 (0.5 μg).
All IVT reactions were scaled to 12.5 µL total volume. Following the
completion of the IVT reaction, the GFP emission was read in a
1.
A. Laroche-Clary, V. L. Morvan, T. Yamori and J. Robert,
Mol. Cancer Ther., 2010, 9, 3315-3321.
B. Rochat, J. M. Morsman, G. I. Murray, W. D. Figg and H.
L. McLeod, J. Pharmacol. Exp. Ther., 2001, 296, 537-541.
F. P. Guengerich, AAPS J, 2006, 8, E101-E111.
R. W. Brueggemeier, J. C. Hackett and E. S. Diaz-Cruz,
Endocr. Rev., 2005, 26, 331-345.
P. Conte and A. Frassoldati, Breast J., 2007, 13, 28-35.
R. Haque, S. A. Ahmed, A. Fisher, C. C. Avila, J. Shi, A. Guo,
2.
3.
4.
5.
6.
Greiner-Bio One 384-well small volume plate on
a Tecan
T. Craig Cheetham and J. E. Schottinger, Cancer Med.
,
SPECTRAFluorPlus Plate Reader with 485 nm excitation and 535 nm
emission filters.
2012, 1, 318-327.
7.
C. H. Van Poznak and D. F. Hayes, J. Natl. Cancer Inst.
,
2006, 98, 1261-1263.
8.
L. R. Ayres, d. A. O. Baldoni, A. d. P. S. Borges and L. R. L.
Pereira, Int. J. Clin. Pharm., 2014, 36, 45-54.
DNA gel electrophoresis
9.
G. Attard, A. S. Belldegrun and J. S. De Bono, BJU Int.
2005, 96, 1241-1246.
,
Compounds were mixed with 40 μg/mL pUC19 plasmid in 10
mM potassium phosphate buffer, pH 7.4. To determine the effect of
light, samples were irradiated with a 470 nm LED for 1 hr. Samples
10.
J. S. de Bono , C. J. Logothetis , A. Molina , K. Fizazi , S.
North , L. Chu , K. N. Chi , R. J. Jones , O. B. J. Goodman , F.
Saad , J. N. Staffurth , P. Mainwaring , S. Harland , T. W.
Flaig , T. E. Hutson , T. Cheng , H. Patterson , J. D.
Hainsworth , C. J. Ryan , C. N. Sternberg , S. L. Ellard , A.
Fléchon , M. Saleh , M. Scholz , E. Efstathiou , A. Zivi , D.
Bianchini , Y. Loriot , N. Chieffo , T. Kheoh , C. M. Haqq
and H. I. Scher N. Engl. J. Med., 2011, 364, 1995-2005.
N. M. DeVore and E. E. Scott, Nature, 2012, 482, 116-119.
were then incubated for 12 hr at 37 °C in the dark. Single and
double-strand DNA break controls were prepared, and the DNA
samples were resolved on agarose gels, as described previously.55
In brief, samples were resolved on a 1% agarose gels prepared
in tris-acetate buffer with 0.3 μg of plasmid/lane. The gels were
stained with 0.5 μg/mL ethidium bromide in tris-acetate buffer at
RT for 40 min, destained with tris-acetate buffer, and imaged on a
ChemiDoc MP System (Bio-Rad).
11.
12.
M. N. Stein, S. Goodin and R. S. DiPaola, Clin. Cancer Res.
2012, 18, 1848-1854.
,
13.
14.
M. C. E. McFadyen, W. T. Melvin and G. I. Murray, Mol.
Cancer Ther., 2004, 3, 363-371.
R. D. Bruno and V. C. O. Njar, Bioorg. Med. Chem., 2007,
15, 5047-5060.
L. K. Nieman, Pituitary, 2002, 5, 77-82.
T. Lynch and A. Price, American family physician, 2007, 76,
391-396.
Z. Bibi, Nutr. Metab., 2008, 5, 1-10.
D. D. Young and A. Deiters, Org. Biomol. Chem., 2007, 5,
999-1005.
L. Zayat, C. Calero, P. Albores, L. Baraldo and R.
Etchenique, J. Am. Chem. Soc., 2003, 125, 882-883.
V. Nikolenko, R. Yuste, L. Zayat, L. M. Baraldo and R.
Etchenique, Chem. Commun., 2005, 13, 1752-1754.
Singlet oxygen assay
Compounds were serially diluted in 96 well plates in
Extracellular Solution (10 mM HEPES pH 7.5, 145 mM NaCl, 10 mM
Glucose, 1.2 mM CaCl2, 1.2 mM MgCl2, 3.3 mM KH2PO4, 0.8 mM
K2HPO4, 50 U/ml Penicillin and 50 mg/ml Streptomycin). To this was
added Singlet Oxygen Sensor Green (SOSG) reagent to give a final
concentration of 5 μM. The plates were read on a SpectraFluor Plus
plate reader with an excitation filter of 485 nm and emission of 535
nm both pre- and post-irradiation with the Loctite Indigo LED for 1
min. The relative values of the SOSG emission were plotted as a
function of compound concentration to give a dose response.
15.
16.
17.
18.
19.
20.
21.
22.
23.
L. Zayat, M. Salierno and R. Etchenique, Inorg. Chem.
2006, 45, 1728-1731.
,
Acknowledgements
K. L. Ciesienski, K. L. Haas, M. G. Dickens, Y. T. Tesema and
K. J. Franz, J. Am. Chem. Soc., 2008, 130, 12246-12247.
K. L. Ciesienski, L. M. Hyman, D. T. Yang, K. L. Haas, M. G.
Dickens, R. J. Holbrook and K. J. Franz, Eur. J. Inorg. Chem.
2010, 2010, 2224-2228.
This work was supported by the National Institutes of Health
(5R01GM107586) and the American Cancer Society (RSG-13-
079-01-CDD). We would like to thank the University of
Kentucky Environmental Research Training Laboratory (ERTL)
for their assistance in some chemical analysis. A. Z. was
,
24.
K. L. Ciesienski and K. J. Franz, Angew. Chem. Int. Ed.
2011, 50, 814-824.
,
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7
Please do not adjust margins

Please do not adjust margins
Dalton Transactions
Page 8 of 9
View Article Online
DOI: 10.1039/C6DT04405K
ARTICLE
Journal Name
25.
T. Respondek, R. N. Garner, M. K. Herroon, I. Podgorski, C. 50.
L. Jia and X. Liu, Curr. Drug Metab., 2007, 8, 822-829.
Turro and J. J. Kodanko, J. Am. Chem. Soc., 2011, 133,
17164-17167.
J. Prakash and J. J. Kodanko, Curr. Opin. Chem. Biol., 2013,
17, 197-203.
T. Respondek, R. Sharma, M. K. Herroon, R. N. Garner, J.
D. Knoll, E. Cueny, C. Turro, I. Podgorski and J. J. Kodanko,
ChemMedChem, 2014, 9, 1306-1315.
51.
P. R. Ortiz de Montellano, Cytochrome P450 : structure,
mechanism, and biochemistry, Kluwer Academic/Plenum
Publishers, New York, 3rd edn., 2005.
26.
27.
52.
53.
S. F. Bellon, J. H. Coleman and S. J. Lippard, Biochemistry
1991, 30, 8026-8035.
,
M. V. Keck and S. J. Lippard, J. Am. Chem. Soc., 1992, 114,
3386-3390.
28.
29.
T. Omura, Chem. Biol. Interact., 2006, 163, 86-93.
M. L. Berger, F. Hammerschmidt, R. Qian, S. Hahner, A.
Schirbel, M. Stichelberger, R. Schibli, J. Yu, V. B. Arion, A.
Woschek, E. Öhler and I. M. Zolle, Mol. Pharm., 2013, 10,
1119-1130.
M. B. Díaz-Valenzuela, S. D. Phillips, M. B. France, M. E.
Gunn and M. L. Clarke, Eur. J. Chem., 2009, 15, 1227-1232.
J. C. Griepenburg, T. L. Rapp, P. J. Carroll, J. Eberwine and
I. J. Dmochowski, Chem. Sci., 2015, 6, 2342-2346.
J. Mosquera, M. I. Sanchez, M. E. Vazquez and J. L.
Mascarenas, Chem. Commun., 2014, 50, 10975-10978.
N. Karaoun and A. K. Renfrew, Chem. Commun., 2015, 51,
14038-14041.
54.
55.
M. V. Keck, J. Chem. Ed., 2000, 77, 1471-1473.
B. S. Howerton, D. K. Heidary and E. C. Glazer, J. Am.
Chem. Soc., 2012, 134, 8324-8327.
E. Wachter, D. K. Heidary, B. S. Howerton, S. Parkin and E.
C. Glazer, Chem. Commun., 2012, 48, 9649-9651.
B. S. Howerton, D. K. Heidary and E. C. Glazer, J Am Chem
Soc, 2012, 134, 8324-8327.
56.
57.
58.
59.
60.
61.
62.
30.
31.
32.
33.
34.
35.
A. N. Hidayatullah, E. Wachter, D. K. Heidary, S. Parkin and
E. C. Glazer, Inorg. Chem., 2014, 53, 10030-10032.
J. D. Knoll, B. A. Albani and C. Turro, Acc. Chem. Res.
,
2015, 48, 2280-2287.
D. K. Heidary and E. C. Glazer, Chembiochem, 2014, 15,
507-511.
E. Wachter, B. S. Howerton, E. C. Hall, S. Parkin and E. C.
Glazer, Chem. Commun., 2014, 50, 311-313.
T. Omura and R. Sato, J. Biol. Chem., 1964, 239, 2379-
2385.
B. A. Albani, C. B. Durr and C. Turro, J. Phys. Chem. A
2013, 117, 13885-13892.
,
B. M. A. van Vugt-Lussenburg, E. Stjernschantz, J.
Lastdrager, C. Oostenbrink, P. E. Vermeulen and J. N. M.
Commandeur, J. Med. Chem., 2007, 50, 455-461.
A. W. Munro, D. G. Leys, K. J. McLean, K. R. Marshall, T.
W. Ost, S. Daff, C. S. Miles, S. K. Chapman, D. A. Lysek, C.
C. Moser, C. C. Page and P. L. Dutton, Trends Biochem.
Sci., 2002, 27, 250-257.
36.
37.
38.
C. R. Jefcoate, Methods Enzymol, 1978, 52, 258-279.
C. W. Locuson, J. M. Hutzler and T. S. Tracy, Drug Metab.
Dispos., 2007, 35, 614-622.
39.
40.
41.
42.
N. A. Hosea, G. P. Miller and F. P. Guengerich, Biochem.
2000, 39, 5929-5939.
Y.-T. Lee, E. C. Glazer, R. F. Wilson, C. D. Stout and D. B.
Goodin, Biochem., 2011, 50, 693-703.
A.-M. A. Hays, A. R. Dunn, R. Chiu, H. B. Gray, C. D. Stout
and D. B. Goodin, J. Mol. Biol., 2004, 344, 455-469.
A. R. Dunn, A.-M. A. Hays, D. B. Goodin, C. D. Stout, R.
Chiu, J. R. Winkler and H. B. Gray, J .Am. Chem. Soc., 2002,
124, 10254-10255.
,
43.
44.
A. R. Dunn, I. J. Dmochowski, A. M. Bilwes, H. B. Gray and
B. R. Crane, Proc. Natl. Acad. Sci. U.S.A., 2001, 98, 12420-
12425.
S. M. Contakes, G. A. Juda, D. B. Langley, N. W. Halpern-
Manners, A. P. Duff, A. R. Dunn, H. B. Gray, D. M. Dooley,
J. M. Guss and H. C. Freeman, Proc. Natl. Acad. Sci. U.S.A.
2005, 102, 13451-13456.
,
45.
46.
47.
B. Durham and F. Millett, BBA-Bioenergetics, 2012, 1817,
567-574.
X.-J. Yang, F. Drepper, B. Wu, W.-H. Sun, W. Haehnel and
C. Janiak, Dalton Trans., 2005, 2, 256-267.
K. Szaciłowski, W. Macyk, A. Drzewiecka-Matuszek, M.
Brindell and G. Stochel, Chem. Rev., 2005, 105, 2647-
2694.
48.
49.
M. D. Burke, S. Thompson, R. J. Weaver, C. R. Wolf and R.
T. Mayer, Biochem. Pharmacol., 1994, 48, 923-936.
N. Goyal, J. Liu, L. Lovings, P. Dupart, S. Taylor, S. Bellow,
L. Mensah, E. McClain, B. Dotson, J. Sridhar, X. Zhang, M.
Zhao and M. Foroozesh, Chem. Res. Toxicol., 2014, 27,
1431-1439.
8 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 9 of 9
Dalton Transactions
G
T
C
C
A
A
G
G
T
C
N
T
A
N
C
G
A
N
G
T
N
N
T
N
N
C
C
OH2
N
Ru
O
A
Ru
A
G
G
T
T
C
C
OH2
N
N
A
G
G
N
T
T
C
A
N
C
G
T
A
O
T
G
C
T
T
A
C
A
G
G
C
A
H
H
O
N
N
FeIII
N
N
S
A light-activatedprodrug delivers both a
DNA damaging metal center and a
cytochrome P450 inhibitor to prevent
drug resistance.