The importance of cellular localisation of probes: Synthesis, photophysical properties, DNA interactions and cellular imaging properties of rhenium dppz complexes with known cellular localisation vectors

Article abstract of DOI:10.1039/c1nj20662a

The synthesis, photophysical properties, DNA binding, DNA cleavage and cellular imaging behaviour of a range of complexes of the type [Re(CO) ₃(dppz)(PyR)]⁺ are reported, where PyR represents a range of substituted pyridines which have previously been studied for cellular localisation of related complexes. Confocal imaging experiments confirm that the complexes retain the variety of cellular localisation behaviour associated with the PyR units in other complexes, and suggest applications as probes for oligonucleotides in specific cellular compartments (e.g. mitochondrial DNA). This study illustrates the importance of considering cellular localisation as a prime consideration in the design of probes for in vivo application. The Royal Society of Chemistry and the Centre National de la Recherche Scientifique.

Full text of DOI:10.1039/c1nj20662a

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New Journal of Chemistry
A journal for new directions in chemistry
www.rsc.org/njc
Volume 36 | Number 1 | January 2012 | Pages 1–180
ISSN 1144-0546
COVER ARTICLE
Coogan, Mishra et al.
The importance of cellular localisation of probes: rhenium dppz complexes
with known cellular localisation vectors

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Cite this: New J. Chem., 2012, 36, 64–72
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PAPER
The importance of cellular localisation of probes: synthesis,
photophysical properties, DNA interactions and cellular imaging
properties of rhenium dppz complexes with known cellular localisation
vectorsw
F. L. Thorp-Greenwood,a M. P. Coogan,*a L. Mishra,*b N. Kumari,b G. Raic and
S. Saripellad
Received (in Montpellier, France) 29th July 2011, Accepted 19th September 2011
DOI: 10.1039/c1nj20662a
The synthesis, photophysical properties, DNA binding, DNA cleavage and cellular imaging
behaviour of a range of complexes of the type [Re(CO)3(dppz)(PyR)]+ are reported, where PyR
represents a range of substituted pyridines which have previously been studied for cellular
localisation of related complexes. Confocal imaging experiments confirm that the complexes retain
the variety of cellular localisation behaviour associated with the PyR units in other complexes,
and suggest applications as probes for oligonucleotides in specific cellular compartments
(e.g. mitochondrial DNA). This study illustrates the importance of considering cellular
localisation as a prime consideration in the design of probes for in vivo application.
vessels,7 while other related complexes8 have been successfully
Introduction
applied in cell imaging after bioconjugation to cell penetrating
The use of large, planar aromatic systems as DNA intercallators
units (e.g. polyarginines). In general, while there are many
is well established in chemical and biological studies of DNA
and related systems, and dipyrido[3,2-a:20,30-c]phenazine (dppz)
studies of tuning the photophysical response of these probes to
DNA/RNA, there has been much less attention paid to the
1 has become a standard ligand in the design of metallointer-
cellular localisation of the probes, although these may be
crucial for in vivo applications. In fact, one study9 has shown
callators. In the field of luminescent dppz complexes for DNA
binding studies, cationic ruthenium complexes of the general
that rather than accumulating in the nucleus, a variety of
formula [Ru(bpy)2(dppz)]2+ have been widely applied and
Ru-dppz complexes showed accumulation in cytoplasmic
studied both experimentally and theoretically due to their strong
organelles, which could limit applicability as in vivo DNA
probes. A recent interesting study10 showed that in a series of
interaction with DNA, and the remarkable photoswitching
upon interaction with DNA.1–5 Due to complex modulation
dialkoxy substituted complexes [Ru(phen)2dppz-(OR)2],
of the nature of the excited states it is possible to achieve up
where R = C2–C6, all the probes showed good uptake, with
to 104 enhancement of emission intensity upon DNA binding
C2 staining only the nucleus, excluding the nucleolus, C4
compared to the free aqueous species.6 While there are many
localising in cytoplasm and nucleoli, and C6 giving intense
studies of in vitro application of such Ru-dppz systems, in vivo
cytoplasmic staining, and weak nucleolar staining. These
application is rarer, due to complications relating to cellular
dramatic differences in localisation with small changes to
uptake and distribution of such species. For instance,
structure illustrate one of the problems with probe design,
[Ru(phen)3]2+ shows a tendency to be trapped in endocytotic
that an alteration in probe structure may change not only its
response to analytes (the complexes in this study had differing
responses to DNA/RNA/mRNA) but also its localisation. In
a Department of Chemistry, Cardiff University, Cardiff,
intensity-based detection this can lead to difficulty in distinguishing
between areas with high probe concentration in the less emissive
form, and those of low concentration in the highly emissive form.
In order to allow meaningful in vivo studies a fuller understanding
of the cellular localisation of probes must be developed. By
comparison to the dominant ruthenium complexes, there has been
less work on the related rhenium complexes, although it was
shown some years ago that Re-dppz complexes were capable of
binding DNA, and the photophysics and electronic basis of the
UK CF10 3AT. E-mail: cooganmp@cf.ac.uk; Fax: 02920874066
b Department of Chemistry, Faculty of Science,
Banaras Hindu University, Varanasi-221 005, India
c Department of Molecular and Human Genetics, Faculty of Science,
Banaras Hindu University, Varanasi-221 005, India
d Department of Biochemistry, Faculty of Science,
Banaras Hindu University, Varanasi-221 005, India
w Electronic supplementary information (ESI) available: further
details of the photophysical properties of complexes 3–8 and full
photophysical data, UV vis and EB titration data, and further cell
images are available. See DOI: 10.1039/c1nj20662a
c
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interaction have been studied in some depth.11–23 In particular,
it has been shown that complexes of the general structure
[Re(CO)3(dppz)(X)] where X represents a wide variety of axial
ligands are very weakly emissive in aqueous media, but show
dramatic emission enhancements in the presence of DNA or
oligonucleotides, ranging from > 10 fold enhancement,12 to a
‘switch on’ from essentially non-emissive,23 depending upon
substitution and conditions. There is a growing body of
literature24 showing that rhenium polypyridyl complexes are
useful in cell imaging experiments, in particular, rhenium
fac-tricarbonyl complexes of chelating bipyridines and related
species have been shown to be advantageous over other triplet
emitters due to the flexibility of having an additional coordination
site once the requirement for emissivity of an [Re(bpy)(CO)3] core
is satisfied. This site is usually occupied by a substituted
pyridine ligand, which can be used as a vector to control cell
uptake and localisation, while the photophysical properties are
largely dominated by the nature of the bipyridine. This
pyridine ligand has been shown to be capable of controlling
uptake and directing localisation to organelles such as
mitochondria, membranes, vacuoles and nucleoli.25 In order
to help in the design of Re-dppz probes, therefore, it was of
interest to investigate the effect of systematically varying the
axial ligand to generate a series of rhenium dppz complexes
which, in principle, could be expected to retain the useful DNA-
related properties but show variations in cellular localisation
and uptake. This is of interest for three reasons: to determine
how/whether variation of the pyridine ligand changes the
interaction with DNA; to determine the optimum design for
the cellular uptake of rhenium dppz species; and to potentially
target DNA in specific cellular compartments.
species is problematical. In the case of the dppz complexes
currently under investigation, again, reaction of the acetonitrile
complex 3 with 3-chloromethylpyridine proved an unfruitful
approach to 8, with only the chloro-complex 9 being isolated as
a discrete product indicating that halide abstraction interferes
with coordination of the pyridine (Scheme 2).
Fortunately it was possible to access the desired chloromethyl
complex 8 by treatment of hydroxymethyl complex 5 with
thionyl chloride, followed by quenching with saturated aqueous
ammonium hexafluorophosphate to isolate the product as the
hexafluorophosphate salt (Scheme 3).
Photophysical studies
As the photophysics of rhenium polypyridyl complexes are
dominated by the nature of the polypyridine ligands, and the
axial ligands typically are less important it was anticipated that
the series of complexes 3–8 would show similar photophysical
properties to each other, and to the range of previously reported
rhenium-dppz complexes, however a brief investigation of these
properties was undertaken to confirm that all of the complexes
conformed to the expected behaviour. This was deemed
especially important in the case of 6 as it has been previously
noted that rhenium polypyridines substituted with alkyl chains
can show anomalous photophysical behaviour as a result of
chain-wrapping events.28 The electronic spectra of complexes
3–8 all show intense absorptions centred around 280 nm,
assigned as ligand-centred 1IL p–p* and n–p* transitions, with
multiple weaker peaks and shoulders at lower energy centred
at around 380 nm assigned as 1MLCT (dp(Re)–p*(dppz))
absorptions by analogy with previous work (see ESI for more
dataw).13 It has previously been noted that the luminescence of
[Re(dppz)Py] species is moderate, and dominated by 3IL
states13 while upon DNA binding12,23 the intensity of emission
increases, although the effect is less pronounced than for related
ruthenium systems. The nature of any substitution of the axial
pyridine ligand typically has little effect upon the emission of such
complexes, and so it was to be expected that complexes 4–8 would
show similar photophysics, while 3 could have been expected to
differ as the replacement of a pyridine ligand by another group in
Re dppz complexes has been reported to modulate the lumines-
cence (at least when the axial ligand is a halide). In fact all of the
complexes 3–8 showed the typical excitation and emission of
[Re(dppz)(Py)] species with excitation maxima around 345 nm
and two emission bands centred at 455 ꢀ 10 nm and 550 ꢀ 5 nm
except 4 in which the high energy band is shifted to 495 nm
(see ESI for more dataw). These photophysical attributes
suggested that complexes 3–8 had potential as imaging agents
and so further studies of their behaviour were undertaken.
Results and discussion
Synthesis of complexes
Given the requirement for a range of rhenium dppz complexes with
varied axial ligands the common precursor [ReBr(CO)3dppz] was
prepared by reaction of [ReBr(CO)5] with dppz 1 (itself prepared
by the method of Barton)26 in toluene at sub-reflux for 18 h to
give the expected product 2 in excellent yield and high purity.
Subsequent activation of the bromide with silver tetrafluoroborate
in acetonitrile gave the derived cationic complex 3 containing
an acetonitrile ligand, which in analogous rhenium bipyridine
chemistry is a common reactive intermediate used to facilitate
the formation of the pyridine substituted complexes
(Scheme 1). In order to generate a range of pyridine-substituted
rhenium dppz complexes 3 was reacted with a number of
3-substituted pyridines under standard conditions (excess
ligand, chloroform sub-reflux, 16 h) to give the derived
complexes 4–7 in 50–80% yields, and the identity of the
complexes was in each case confirmed by NMR, IR and mass
spectrometry. The pyridine ligands were chosen as they had
previously been shown in the analogous bipyridine series to give
a range of degrees of cellular uptake and localisation pattern.
One complex which is attractive as in the bipyridine series it
shows mitochondrial localisation27 is the chloromethypyridine
complex 8, however previous work had indicated that the direct
coordination of 3-chloromethypyridine to activated rhenium
DNA binding studies
While it is well established that simple rhenium dppz complexes are
strong DNA binders, it was important to confirm that the inclusion
of large units such as in the myristyl or cyclohexyl derived
complexes (6 and 7 respectively) had not precluded DNA binding.
UV-vis titrations
Electronic absorption spectroscopy is an effective tool to
examine the binding mode of DNA with metal complexes.29
c
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Scheme 1 Synthesis of complexes 3–7.
the changes in their absorbance with increasing concentration
of DNA. A representative spectrum of complex 6 is shown in
Fig. 1 (see ESI for more dataw). The value of Kb is obtained as
a ratio of slope to intercept obtained from the plot of [DNA]/
(ea ꢁ ef) vs. [DNA], and are summarised in Table 1. Complexes
3–8 all show values in the region of 0.7 ꢁ 6 ꢂ 105 Mꢁ1 which is
comparable with the values previously observed for related
species,11,13 and indicative of strong binding to DNA. The Kb
values thus obtained varied in the order Kb (6) > Kb (5) c Kb
(4) > Kb (3) > Kb (7) > Kb (8), with 5 and 6 being
significantly higher than the other complexes. The anomalously
strong DNA binding observed with complexes 5 and 6 could
be explained by hydrogen bonding from the hydroxymethyl
pyridine of 5 to a DNA base and by hydrophobic interaction
between the myristyl chain of 6 with DNA. Hydrophobic
effects are important in DNA binding33 and the enthalpic gain
of removing a C13 chain from the aqueous medium may well
be important to the high binding constant observed with this
complex. It should be noted that the acetonitrile ligand of 3
is regarded as labile and it is presumed that the species
equilibrates between aqua- and halo- complexes in buffered
media, with the final species being likely to be coordinated at
metal by a group from the DNA fragment itself. That the
binding constant of this complex is not significantly higher
than species which lack a labile ligand suggest that any
such interaction is weak, as may be expected for phosphate
oxygens, or even the amidic nitrogens of DNA bases.
Scheme 2 Attempted synthesis of chloromethyl complex 8.
Scheme 3 Synthesis of chloromethyl complex 8.
The predominant target of metal-based anticancer complexes
is often believed to be binding to DNA through N-7 of
guanine.30 Intercalative binding of a complex with DNA is
accompanied by both a hypochromic and bathochromic shift
in the absorption spectrum.31 Intercalation can occur due to a
strong stacking interaction between the aromatic rings present
in the ligand framework and the DNA base pairs, and if a
p* orbital of the intercalated ligand can couple with a p orbital
of the DNA base pairs, the p–p* transition energy decreases
inducing a red shift in the observed band, whereas, if a ligand
p orbital is partially filled, it decreases the probability and
concomitantly results in hypochromism.32 Thus, in order to
provide evidence for the possibility of binding of each complex
to CT-DNA, spectroscopic titration of solutions of each of the
complexes with CT-DNA was performed. In order to compare
the binding strength of complexes with CT DNA, the intrinsic
binding constant Kb was calculated in each case by monitoring
Competitive binding with ethidium bromide (EB)
The ability of a complex to affect the fluorescence intensity of
EB-DNA adduct is a reliable tool for the measurement of its
affinity towards DNA, irrespective of its mode of binding.
Intense fluorescence is observed from EB in the presence of
DNA, owing to its strong intercalation between adjacent DNA
base pairs. A complex binds with DNA by the displacement of
the EB which is already bound to DNA. Consequently, the
intensity of emission is reduced as emission from of free EB is
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Fig. 1 UV-vis absorption spectra of (a) [Complex 3] = 10 mM in the absence and in presence of increasing amounts of DNA = 0–100 mM. (b)
[Complex 6] = 10 mM in the absence and in presence of increasing amounts of DNA = 0–100 mM. Arrow shows the absorbance changes upon
increasing DNA concentration.
The Stern–Volmer constant (Ksv) is evaluated as 1.3, 1.0, 2.0,
Table 1 Kb values for complexes 3–8
5.5, 0.86 & 0.61 for complexes 3, 4, 5, 6, 7, and 8 respectively
which gives the order of the observed extent of ethidium
bromide displacement by the complexes as 6 > 5 > 3 >
4 > 7 > 8. This is the same gross order as seen in the uv-vis
work but with 3 and 4 switching places, presumably as a result
of their different interactions with EB complicating the simple
DNA affinity relationship seen in the UV-vis titrations.
In summary, all complexes 3–8 showed strong DNA binding
with KB in the region of 104–105 Mꢁ1 which are in the
typical range for rhenium dppz complexes and bode well for
applications as cellular probes for DNA.
Complex
Kb/105 (Mꢁ1
3
1.17
4
2.56
5
5.59
6
6.28
7
0.938
8
0.728
)
readily quenched by surrounding water molecules.34 Representative
emission spectra of the EB-DNA system in the presence and
absence of complexes 3 and 6 are shown in Fig. 2 (see ESI
for more data). The addition of complexes to DNA pretreated
with EB results in appreciable reductions in emission intensity,
indicative of DNA binding by the complex, and subsequent
aqueous quenching of displaced EB. On the addition of 4–7 mM
of complexes to 10 mM of CT DNA pretreated with EB, B80%
displacement of ethidium bromide was observed. Corresponding
quenching plots of Io/I vs. [Complex]/[DNA] (the inset of Fig. 2)
are in good agreement with the linear Stern–Volmer equation.
DNA cleavage studies
Binding of transition metal complexes with DNA plays an
important role in identification of target structures for
Fig. 2 Emission spectra of EB bound to DNA in the absence and in the presence of, [complex 3 and 6] 0–5 mM, [EB] 10 mM, [DNA] 10 mM. Arrow
shows changes in the emission intensity upon addition of increasing concentration of the complex.
c
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chemotherapeutic agents based on DNA cleavage. However,
even in cases where binding is strong due to a motif such as
dppz, DNA cleavage abilities show additional dependency on
the other substituents present in the complexes. The DNA-
cleaving ability of complexes 3–8 has been studied by the
relaxation of supercoiled pBR322 DNA to the nicked circular
DNA. When circular plasmid DNA is subjected to gel electro-
phoretic mobility assays, relatively fast migration is observed
for the intact supercoiled form (S). However, if scission of
DNA occurs at one strand (nicking), the supercoiled DNA
relaxes to generate a slower-moving open/nicked circular form
(NC). If both strands are cleaved, a linear form (L) is
generated.35 To assess the DNA cleavage ability of the
complexes, supercoiled pBR322 DNA (300 ng mLꢁ1) was
incubated separately with 100 mM of the complexes in 5 mM
Tris-HCl/50 mM NaCl buffer at pH 8.0 (Fig. 3). As compared
to the untreated plasmid DNA sample, there was no change in
the intensities of SC and NC forms of DNA when incubated
with complexes in aqueous buffered solution up to 24 h,
indicating little or no cleavage of DNA by any of the
complexes studied.
Fig. 4 UV light induced cleavage of supercoiled pBR322 plasmid
DNA in the presence of increasing concentration of complexes in
aqueous buffered solution. The plasmid DNA (300 ng mLꢁ1) + complex
mix were incubated for 10 min, followed by UV exposure at 312 nm
(64 W) for 10 min. The samples were then subjected to 1.5% agarose gel
electrophoresis and bands were developed by ethidium bromide staining.
(a) Lane 1, DNA control; Lane 2, DNA + 20 mM of complex 3; Lane 3,
DNA + 40 mM of complex 3; Lane 4, DNA + 60 mM of complex 3;
Lane 5, DNA + 80 mM of complex 3 (b) Lane 1, DNA control; Lane 2,
DNA + 20 mM of complex 5; Lane 3, DNA + 40 mM of complex 5;
Lane 4, DNA + 60 mM of complex 5; Lane 5, DNA + 80 mM of
complex 5 (c) Lane 1, DNA control; Lane 2, DNA + 10 mM of complex
6; Lane 3, DNA + 20 mM of complex 6; Lane 4, DNA + 30 mM of
complex 6; Lane 5, DNA + 40 mM of complex 6; Lane 5, DNA +
40 mM of complex 6; Lane 6, DNA + 50 mM of complex 6.
As no DNA cleavage was observed with complexes 3–8
under ambient conditions, attempts were made to investigate
the photonuclease activity of the complexes. Thus each
complex exposed to UV light at 312 nm (64 W) for 10 min
in an aqueous solution containing DNA. All complexes except
7 and 8 cause a linear increase in the intensity of NC DNA
band accompanied with the similar decline in SC DNA in a
concentration dependent manner in the aqueous buffer.
Complex 6 converted more than 80% of SC form into NC
form at a concentration of 50 mM, whereas 80 mM of 3 and 5 is
required for similar conversion (Fig. 4). DNA cleavage by
complex 6 is significantly higher than that observed with
complex 3, which could be related to the electron releasing
substituent in view of an earlier report.36 However, DNA cleavage
by complex 3 could be related to the liability of the acetonitrile
ligand which provides an opportunity for coordination of
rhenium by a DNA base, usually guanine.37 It should be noted
that the significantly higher DNA binding ability of complex 6
compared to complexes 3 and 5 may also be significant in their
relative cleavage efficiencies.
do not cleave DNA under physiological conditions, and show
attractive photophysical properties for cell imaging, they were
considered potential candidates for live cell imaging studies.
Cell imaging studies
To the best of our knowledge, while their photophysical
properties have been widely studied, there are no previous
reports of the application of rhenium dppz complexes in
fluorescent cell imaging. Complexes 3, 5, 6 and 9 were selected
for cell imaging studies due to their favourable binding and
fluorescence properties and the known uptake and localisation
behaviour of the bipyridine24–28 and (in a recent example
involving polylactide chains) phenanthroline38 analogues,
which have been examined in detail and confirmed by
co-localisation experiments with commercial stains. Given
the interest in this case of comparing the known tendency of
dppz complexes to show nuclear localisation, and the emission
enhancements observed upon binding these complexes with
DNA the imaging experiments were undertaken under
colocalisation conditions with Hoechst34 580 a known nuclear
and DNA specific stain. By comparing the localization of the
new complexes with this material, and by comparing the intensity
of rhenium-based emission from the nucleus (which is DNA rich
and thus shows intensity-enhanced Re-based emission) with the
intensity of other areas the possibility of binding to DNA/RNA
present in other organelles can be examined. It should be noted
that in addition to the luminescence enhancements reported for
In summary, there is some interesting and axial ligand-
dependent DNA photocleavage behaviour associated with
certain examples of the rhenium dppz complexes studied in
this report which will be the focus of further studies. However,
none of the complexes studied showed DNA cleavage in the
absence of irradiation. Thus, as all complexes bind DNA well,
Fig. 3 Ethidium bromide stained agarose gel (1.5%) of pBR322
plasmid DNA (300 ng mLꢁ1) in the presence of 100 mM complexes
after 24 h of incubation, Lane 1: DNA control. Lane 2: pBR322 + 3.
Lane 3: pBR322 + 4. Lane 4: pBR322 + 5. Lane 5: pBR322 + 6.
Lane 6: pBR322 + 7. Lane 7: pBR322 + 8.
c
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rhenium dppz complexes in the presence of DNA, other
environments can cause enhanced emission in comparison to
aqueous solution. Changing from water to organic solvents
can cause many-fold enhancements of emission intensity23
and it is likely that the complexes in question could show
interaction with, and subsequent luminescent enhancement in
e.g. bilipid membranes. In the event of the complexes localising
in aqueous compartments of the cell it is unlikely that they
would be detected-the emission of aqueous solutions of such
species being barely detectable by fluorimetry, and certainly
below the detection limits of the microscope. Thus, it is
impossible to be sure of the full localisation of these complexes
as in certain areas they will be difficult to detect. However, as
they all have the typical photophysics of rhenium dppz
complexes and show the same enhancements in the presence
of DNA, lipids etc., any differences observed in patterns of
emission intensity between the complexes must indicate
control of localisation. Thus differences observed between the
localisation of these complexes would indicate that using dppz
complexes to map cellular DNA/RNA without prior consideration
of their localisation preferences may be misleading. Incubation of
the complexes with HeLa cells followed by examination by laser
scanning confocal microscopy indicated that the complexes were all
taken up by cells and showed different patterns of localisation,
validating the theory that the choice of axial ligand is vital to
the application of rhenium dppz DNA probes in vivo, and
could provide probes selective for e.g. mitochondrial DNA.
The acetonitrile complex 3 showed (Fig. 5, 3 shown in green)
very similar localisation to that of the standard Hoechst stain
(shown in blue), indicating localisation in DNA rich sections
of the nucleus from the characteristic emission pattern from
the centre of the cell (as indicated by overlap with brightfield
images) and overlap with the Hoechst dye (turquoise). As has
been previously noted this complex is highly reactive and it is
likely that the acetonitrile has been displaced by biological
material during the incubation, so the localisation is probably
dominated by lipophilicity and DNA binding of the dppz
ligand, explaining the nuclear localisation. Complex 5, the
hydroxymethyl pyridine complex showed (Fig. 6) a slightly
different pattern of localisation, with the nuclear localisation
observed for the Hoechst stain and 3 being supplemented by
some localisation in the perinuclear region of the cytoplasm,
apparently concentrated in organelles. As we have previously
noted that rhenium hydroxymethylpyridine complexes show
some degree of localisation in mitochondria,25 is likely that the
extra-nuclear regions in which 5 shows intense staining are
mitochondria, and from the similar relative intensity of the
emission from these regions and from the nucleus (where 5 is
certain to be DNA bound) that in these regions 5 is bound to
mitochondrial DNA. Confirmation of this assumption came
with the imaging observed with complex 8 which bears a
chloromethylpyridine unit, known to strongly direct localisation
almost exclusively to mitochondria by reaction of the
chloromethyl unit with the high concentration of reduced
thiols located in these organelles.27 In this case (Fig. 7) the
emission was almost entirely observed in the perinuclear
region from localisation in mitochondria, and only a small
degree of overlay was observed with the Hoechst nuclear stain,
but again the relative intensities of rhenium-derived emission
from mitochondrial and nuclear regions indicates that the
mitochondrial-localised dye is DNA-bound. Finally, the myristyl
derived complex 6 showed (Fig. 8) much more generalised
cytoplasmic staining with little nuclear staining and more
diffuse patterns of localisation in the cytoplasm than
complexes 5 and 8, clearly localising in a range of organelles and
membrane structures (as is expected from previous experiments
with this ligand). The intensity of the emission from some
organelles and regions is clearly much less than that observed
in the cases where 6 overlaps with Hoechst, indicating that
these regions are not DNA rich so there is no enhancement of
emission. We are confident that the alternative explanation,
that overall levels of uptake of this material are much lower
Fig. 5 Cell imaging with acetonitrile complex 3 (overlaid 3 green
Hoechst Blue) (further data in the ESIw).
Fig. 6 Cell imaging with hydroxymethyl complex 5 (overlaid 5 green
Hoechst Blue) (further data in the ESIw).
c
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New J. Chem., 2012, 36, 64–72 69

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vectors with DNA responsive dppz units. However, crucially
this study demonstrates that cellular localisation parameters
are a vital consideration when designing probes to be applied
in vivo. In the case of probes which rely upon changes in
emission intensity in the presence of an analyte for detection,
and have no ratiometric aspects,39,40 only a full understanding
of the cellular distribution patterns of the probe can allow even
qualitative conclusions to be drawn about relative analyte
distribution in the organisms studied. The patterns of localisation
of metallolumophores are only just becoming understood, as
has been highlighted in a recent review,41 and, currently, the
examples of vectors for controlling the localisation of rhenium
complexes studied in this work represent the only reliable
examples in the literature. Until localisation of metallolumo-
phores is better understood and more predictable, therefore,
great caution must be applied in the interpretation of the results
of imaging experiments relying upon emission intensity to
indicate the presence/absence of analytes.
Experimental
Fig. 7 Imaging with chloromethyl complex 8 (further data in the
ESIw).
Synthesis of complexes
[Re(CO)3(dppz)(MeCN)][BF4] 313. Complex 238 (400 mg,
0.63 mmol) was dissolved in MeCN (2 ml) and AgBF4 (147 mg,
0.759 mmol) was added. The solution was stirred at sub-reflux
in a dark, nitrogen environment overnight. The yellow
solution was filtered and evaporated until a minimum amount
of solvent remained. The compound was precipitated with
ether and was filtered to give the pure product as a yellow solid
(288 mg, 42%). 1H NMR (CD3CN) 9.58 (2H, dd, J = 8.3, 1.4)
9.51 (2H, dd, J = 5.3, 1.4) 8.27 (2H dd, J = 6.5, 3.5) 8.15 (2H
dd, J = 8.3, 5.3) 8.07 (2H dd, J = 6.6, 3.4) 1.98 (3H, s).
13C NMR (CD3CN) 155.6, 149.0, 142.1, 138.4, 136.0, 132.4,
129.9, 129.1, 127.7, 122.5, 2.6. lmax 361, 380 nm.13
[Re(CO)3(dppz)(Py)][BF4] 4. Complex 3 (46 mg, 0.068 mmol)
was dissolved in CHCl3 and pyridine (0.027 ml, 0.34 mmol)
was added. The solution was heated at sub-reflux overnight in
a nitrogen environment. The solution was evaporated to a
minimum volume and ether was added to precipitate the
yellow product (27 mg, 56%).
1H NMR (CHCl3) 9.86 (2H, d, J = 8.3), 9.70 (2H, d,
J = 5.2), 9.45–8.38 (4H, m), 8.29 (2H, dd, J = 5.3, 2.9),
8.16-8.12 (2H, m), 7.80 (1H, t, J = 7.0), 7.28 (2H, t, J = 7.0);
13C NMR (CD3CN) 155.4, 151.8, 148.8, 142.2, 139.6, 138.6,
136.4, 132.4, 130.2, 129.2, 128.3, 126.4. lmax 360, 379 nm nmax
IR (nujol) 2035 (CO), 1947 (CO), 1909 (CO) cmꢁ1. MS (ES+)
[M+H] 632.11; HRMS C26H15N5O3Re calc: 630.0699;
observed: 630.0704.
Fig. 8 Imaging with myristyl complex 6 (further data in the ESIw).
than the other complexes, is not applicable as previous work
demonstrates that the additional lipophilicity associated with
the myristyl chain over simple pyridines leads to much higher
levels of uptake.25,27
[Re(CO)3(dppz)(3-CH2OH-Py)][BF4] 5. Complex 3 (150 mg,
0.22 mmol) was dissolved in CHCl3 and 3-hydroxymethyl-
pyridine (26 mL, 0.27 mmol) was added. The solution was
heated at sub-reflux overnight in a nitrogen environment. The
solution was evaporated to a minimum volume and ether was
added to precipitate the yellow product (138 mg, 84%).
1H NMR (MeOD) 9.97 (2H, d), 9.83 (2H, dd, J = 3.9, 1.4),
8.49–8.35 (6H, m), 8.16–8.11 (2H, m), 7.79 (1H, d, J = 8.4),
7.31 (1H, dd, J = 5.5, 2.2) 13C NMR (CD3CN) 155.2, 150.1,
Conclusions
From the studies detailed herein, it is clear that rhenium dppz
complexes appended through the axial pyridine ligand with
groups known to direct cellular localisation retain both their
useful DNA binding and photophysical properties, while
gaining the organelle specific-localisation desired. The imaging
studies suggest that such complexes may be used as probes for
e.g. mitochondrial DNA by combining organelle targeting
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70 New J. Chem., 2012, 36, 64–72
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149.5, 148.8, 142.3, 140.6, 138.6, 137.4, 136.6, 132.3, 130.2,
129.2, 128.1, 125.8, 59.8. lmax 362, 380 nm. nmax IR (Nujol)
2032 (CO), 1929 (CO), 1915 (CO) cmꢁ1. MS (ES+) [M+H]
662.08; HRMS C27 H17 N5 O4 Re: calc: 660.0805; observed:
660.0811.
DNA binding studies
Absorption titration. The binding of the complexes 3–8 with
DNA was measured in a Na-phosphate buffer solution
(pH 7.2). The absorption ratio at 260 nm and 280 nm of CT
DNA solutions was found as 1.9 : 1. It shows that DNA is
sufficiently free from protein. The concentration of DNA was
then determined by UV/visible absorbance using the molar
absorption coefficient (6600 Mꢁ1 cmꢁ1) at 260 nm.42 The
absorption titrations of 3–8 (10 mM) in Na-phosphate buffer
(pH 7.2) containing 2% acetonitrile against CT DNA were
performed by monitoring the changes in their absorption
spectra. The titration experiments were performed by
maintaining the concentration of metal complexes constant
at 10 mM while the concentration of CT DNA was varied
within 0–100 mM. An equal quantity of CT DNA was also
added to the reference solution to eliminate the absorption by
DNA. From the absorption data, the intrinsic binding
constant Kb was calculated from a plot of [DNA]/(ea ꢁ ef)
vs. [DNA] using the equation:
[Re(CO)3(dppz)(Py-3-O2C(CH2)12CH3)][BF4] 6. Complex 3
(45 mg, 0.0661 mmol) was dissolved in CHCl3 and 3-hydroxy-
methylpyridyl myristate (20 mg, 0.0627 mmol)28 was added.
The solution was heated at sub-reflux overnight in a nitrogen
environment. The solution was evaporated to a minimum
volume and ether was added to precipitate the yellow product
(33 mg, 52%).
1H NMR (CD3CN) 9.77 (2H, d, J = 8.3), 9.59 (2H, d,
J = 5.2), 8.35–8.28 (4H, m), 8.18–8.15 (2H, m), 8.05–8.01
(2H, m), 7.66 (1H, d, J = 7.8), 7.16 (1H, pseudo-t) 4.74 (2H, s)
1.95(2H, m), 1.18 (14H, bs), 0.93 (4H, bs), 0.81 (3H, m). 13C
NMR (CD3CN) 155.2, 155.1 152.0, 151.2 142.2 140.3 138.6
136.4 135.1 132.2 130.3 129.1 128.9 128.0 125.9 53.8 31.1
28.8–28.7 (multiple peaks) 28.5 28.2 21.8 12.9. lmax 360,
[DNA]/(ea ꢁ ef) = [DNA]/(eb ꢁ ef) + [Kb(eb ꢁ ef)]ꢁ1
379 nm. nmaxIR (Nujol) 2035 (CO), 1921 (2 ꢂ CO) cmꢁ1
.
MS (ES+) [M+H] 872.29. HRMS calc: 870.2788; observed:
870.2810.
Where, [DNA] represents the concentration of DNA in base
pairs. The apparent absorption coefficients ea, ef and eb
correspond to Aobsd./[Re], the extinction coefficient for the
free rhenium(I) complex and extinction coefficient for the
rhenium(I) complex in the fully bound form, respectively.43
Kb is the ratio of slope to the intercept.
[Re(CO)3(dppz)(Py-3-CO2Cy)][BF4] 7. Complex 3 (40 mg,
0.0588 mmol) was dissolved in CHCl3 and cyclohexylnicotinate25
(24 mg, 0.118 mmol) was added. The solution was heated at
sub-reflux overnight in a nitrogen environment. The solution
was evaporated to a minimum volume and ether was added to
precipitate the yellow product (26 mg, 52%).
Competitive binding of complexes with ethidium bromide.
Relative binding of the rhenium complexes to CT DNA was
also studied by fluorescence spectroscopy using ethidium
bromide (EB) bound to CT DNA in a Na phosphate buffer
solution (pH 7.2). Competitive binding of Re(I) complexes to
CT DNA resulted in the displacement of bound EB which
subsequently quenches the fluorescence. In a typical experi-
ment, 20 mL of CT-DNA solution (A260 = 2.0) was added to
2.0 mL of EB buffer solution (pH 7.2) and the fluorescence
intensity was measured at excitation wavelength 510 nm and
maximum emission was observed at 600 nm. Aliquots of a
0.1 mM solution of the complexes were then added separately
to DNA-EB solution and resulting fluorescence spectra were
measured after each addition until it reached to maximum
reduction in fluorescence intensity. Stern–Volmer quenching
constants were calculated using the given equation,44
1H NMR (CD3CN) 9.79 (2H, d, J = 7.1), 9.60 (2H, d,
J = 4.1), 8.54 (2H, d, J = 4.9), 8.46 (1H, d, J = 1.7), 8.34
(2H, dd, J = 3.5, 3.4), 8.18 (2H, m), 8.04 (2H, dd, J =
3.4, 3.5), 7.30 (1H, dd, J = 5.6, 2.4), 4.73 (1H, bm), 1.66–1.23
(10H, bm). 13C NMR (CD3CN) 180.1, 161.7, 155.5, 155.3,
151.4, 149.1, 142.5, 139.8, 138.8, 136.7, 132.5, 130.7, 129.3,
128.3, 126.4, 74.0, 30.5, 24.5, 22.7. lmax 363, 380 nm. nmaxIR
(MeCN) 2031 (CO), 1932 (CO), 1911 (CO), 1718 (COOR) cmꢁ1
.
MS (ES+) [M]+ 758.14; HRMS C33 H25 N5 O5 Re: calc:
756.1380; observed: 756.1381.
[Re(CO)3(dppz)(3-CH2Cl-Py)][PF6] 8. Complex 5 (24 mg,
0.0321 mmol) was dissolved in thionyl chloride (1 ml) and the
mixture stirred at room temperature for 2 h. The reaction
mixture was then evaporated to drynes, redissolved in the
minimum volume of acetonitrile, and treated dropwise with a
saturated aqueous solution of ammonium hexafluorphosphate.
The precipitate was collected by filtration, washed with water
and diethyl ether and dried to give the title compound as yellow
crystals (17 mg, 64%).
1H NMR (CD3CN) 9.92 (2H, d, J = 7.4) 9.72 (2H, d,
J = 4.3) 8.48 (2H, dd, J = 6.6, 3.4) 8.43 (1H br. s) 8.30
(3H, m) 8.16 (2H, dd, J = 6.6, 3.4) 7.83 (1H, d, J = 7.4) 7.26
(1H, dd, J = 7.9, 5.6) 4.50 (2H, s). This complex was
insufficiently soluble to obtain meaningful 13C data. lmax
363, 380 nm. nmax IR 2034 (CQO) 1923 (CQO) cmꢁ1. MS
(ES+) [M]+ 680.04; HRMS C27H16ClN5O3Re: calc:
678.0466; observed: 678.0471.
Io/I = 1 + Ksvr,
where Io and I are the fluorescence intensities in absence and
presence of complexes, respectively, Ksv is a linear Stern–
Volmer quenching constant and r is the ratio of the total
concentration of complex to that of DNA. The value of Ksv is
given by the ratio of slope to intercept in a plot of Io/I versus
[Complex]/[DNA].
DNA cleavage. The nuclease activity of the Re(I) complexes
was studied using supercoiled pBR322 plasmid DNA.
Electrophoresis in native agarose gel was used to quantify
the unwinding induced by Re(I) complexes in pBR322,
monitoring the changes in the degree of supercoiling of
pBR322. The cleavage reactions were carried out in a total
c
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New J. Chem., 2012, 36, 64–72 71

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volume of 25 mL containing pBR322 DNA (300 ng mLꢁ1) and
different concentrations of complexes prepared in 5 mM
Tris–HCl buffer (pH 7.2) containing 50 mM NaCl and 0.02%
acetonitrile for 24 h at 37 1C. The samples were analyzed by
electrophoresis for 3 h at 50 V on 1.5% agarose gel in 1 ꢂ TAE
buffer (40 mM Tris acetate and 1 mM EDTA) pH 8.0. The gel
was stained with a 0.5 mg mlꢁ1 ethidium bromide and visualized
by UV light and then photographed for analysis. The extent of
cleavage was measured from the intensities of the bands using
the AlphaImager 2200 software.45
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Cell imaging. HeLa cells, obtained from NCCS (National
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supplemented with 10% FBS (Fetal Bovine Serum) and 5%
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1
ꢂ
PBS and then incubated with Hoechst34 580
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Acknowledgements
We thank the EPSRC Mass Spectrometry National Service
Centre, Swansea for invaluable assistance in obtaining mass
spectra and high resolution mass spectra on the difficult and
labile complexes 4–8.
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