Optimising the synthesis, polymer membrane encapsulation and photoreduction performance of Ru(II)- and Ir(III)-bis(terpyridine) cytochrome c bioconjugates

Article abstract of DOI:10.1039/c3ob40620b

Ruthenium(ii) and iridium(iii) bis(terpyridine) complexes were prepared with maleimide functionalities in order to site-specifically modify yeast iso-1 cytochrome c possessing a single cysteine residue available for modification (CYS102). Single X-ray crystal structures were solved for aniline and maleimide Ru(ii) 3 and Ru(ii) 4, respectively, providing detailed structural detail of the complexes. Light-activated bioconjugates prepared from Ru(ii) 4 in the presence of tris(2-carboxyethyl)-phosphine (TCEP) significantly improved yields from 6% to 27%. Photoinduced electron transfer studies of Ru(ii)-cyt c in bulk solution and polymer membrane encapsulated specimens were performed using EDTA as a sacrificial electron donor. It was found that membrane encapsulation of Ru(ii)-cyt c in PS₁₄₀-b-PAA₄₈ resulted in a quantum efficiency of 1.1 ± 0.3 × 10^-3, which was a two-fold increase relative to the bulk. Moreover, Ir(iii)-cyt c bioconjugates showed a quantum efficiency of 3.8 ± 1.9 × 10^-1, equivalent to a ～640-fold increase relative to bulk Ru(ii)-cyt c.

Full text of DOI:10.1039/c3ob40620b

Organic&
BiomolecularChemistry
www.rsc.org/obc
Volume 11 | Number 28 | 28 July 2013 | Pages 4561–4728
ISSN 1477-0520
PAPER
Pall Thordarson et al.
Optimising the synthesis, polymer membrane encapsulation and photoreduction
performance of Ru(II)- and Ir(III)-bis(terpyridine) cytochrome c bioconjugates

Organic &
Biomolecular Chemistry
View Article Online
View Journal | View Issue
PAPER
Optimising the synthesis, polymer membrane
encapsulation and photoreduction performance
of Ru(II)- and Ir(III)-bis(terpyridine) cytochrome c
bioconjugates†
Cite this: Org. Biomol. Chem., 2013, 11,
4602
David Hvasanov,a Alexander F. Mason,a Daniel C. Goldstein,a Mohan Bhadbhadeb
and Pall Thordarson*a
Ruthenium(II) and iridium(III) bis(terpyridine) complexes were prepared with maleimide functionalities in
order to site-specifically modify yeast iso-1 cytochrome c possessing a single cysteine residue available for
modification (CYS102). Single X-ray crystal structures were solved for aniline and maleimide Ru(II) 3 and
Ru(II) 4, respectively, providing detailed structural detail of the complexes. Light-activated bioconjugates
prepared from Ru(II) 4 in the presence of tris(2-carboxyethyl)-phosphine (TCEP) significantly improved
yields from 6% to 27%. Photoinduced electron transfer studies of Ru(II)–cyt c in bulk solution and
polymer membrane encapsulated specimens were performed using EDTA as a sacrificial electron donor.
It was found that membrane encapsulation of Ru(II)–cyt c in PS140-b-PAA48 resulted in a quantum
efficiency of 1.1 0.3 × 10−3, which was a two-fold increase relative to the bulk. Moreover, Ir(III)–cyt c bio-
conjugates showed a quantum efficiency of 3.8 1.9 × 10−1, equivalent to a ∼640-fold increase relative
to bulk Ru(II)–cyt c.
Received 28th March 2013,
Accepted 17th May 2013
DOI: 10.1039/c3ob40620b
www.rsc.org/obc
available but important proteins, (ii) the excitation wavelength
of the chromophores used should be easily tuneable and (iii)
Introduction
Scientists have endeavoured to understand and exploit the the bioconjugates should be readily incorporated in mem-
modification of proteins by introducing synthetic ligands in branes allowing us to mimic Nature’s ability to compartmenta-
order to enhance or introduce novel functionalities in the field lise protein-based processes. To this end, we have mainly
of bioconjugation chemistry.1 This has led to the development focused on systems based on the readily available redox
of biotechnological, biomedical and pharmaceutical industry enzyme iso-1 cytochrome c (cyt c) derived from Saccharomyces
related devices such as biosensors,2 bioelectronics,3 biofuel cerevisiae which is then activated by light via room temperature
cells4 and bioconjugated therapeutic proteins/drugs.5
photoinduced electron transfer.6,7
In our group, we have studied light-activated bioconjugates
The light-harvesting component using tris(bipyridine)ruthe-
based on redox enzymes as potential building blocks for appli- nium(II) metal complexes with cytochrome c have been exten-
cations ranging from hybrid solar-biofuel cells to biosensors sively studied due to their long fluorescent lifetimes and high
and light-driven nanoreactors. In making these bioconjugates quantum yields.8–10 However, in recent decades, metal com-
we are guided by three design principles: (i) the bioconjugate plexes based on ruthenium(II) or iridium(III) bis(terpyridine)
synthesis should be specific but straightforward using readily complexes have been receiving considerable attention due to
their interesting photophysical, electrochemical and photo-
chemical properties.11 Moreover, bis(terpyridine) complexes
aSchool of Chemistry, University of New South Wales, Sydney, NSW 2052, Australia.
are synthetically straightforward as the use of 4′-functionalised
E-mail: p.thordarson@unsw.edu.au; Fax: +61-2-9385-6141; Tel: +61-2-9385-4478
ligands avoids complications in chirality and allows simplified
synthesis of heteroleptic complexes. This has already allowed
us to synthesise several bis(terpyridine)ruthenium(II) com-
plexes and bis(terpyridine)iridium(III)12 complexes that can be
excited at around 480 nm and 370 nm, respectively.
bAnalytical Centre, University of New South Wales, Sydney, NSW 2052, Australia
†Electronic supplementary information (ESI) available: Terpyridine ligand syn-
thesis, mono-complex synthesis, in-gel tryptic digest, photoreduction and
quantum efficiency calculations, Ru(II)–cyt
c bioconjugate structural model,
UV-Vis/MALDI-TOF spectra of bioconjugates, 1H/13C NMR spectra for novel com-
pounds and selective crystallographic distances and angles. CCDC 931062 and
931063. For ESI and crystallographic data in CIF or other electronic format see
DOI: 10.1039/c3ob40620b
Electron transfer studies involving cytochrome c have been
reported in literature, predominantly using tris(bipyridine)-
4602 | Org. Biomol. Chem., 2013, 11, 4602–4612
This journal is © The Royal Society of Chemistry 2013

View Article Online
Organic & Biomolecular Chemistry
Paper
ruthenium(II) complexes via functionalisation of histidine,13
lysine14 or cysteine residues.15 In contrast, electron transfer
based on bis(terpyridine)ruthenium(II) complexes as electron
donors have been limited. Hamachi et al.16 had reported room
temperature electron transfer in apomyoglobin reconstituted
with ruthenium bis(terpyridine) complex appended heme as
the enzyme cofactor. Additionally, our group have previously
reported light-activated bioconjugates involving iso-1 cyto-
chrome c capable of electron transfer based on short and long
chain spacer bis(terpyridine)ruthenium(II) complexes.
Herein, we describe a novel approach for improving the syn-
thesis of light-activated ruthenium(II) and iridium(III) based
cytochrome c bioconjugates including relevant single crystal
structures of ruthenium(II) complexes Ru(II) 3 and Ru(II) 4. We
also demonstrate the membrane encapsulation of Ru(II)–cyt c
bioconjugates in polystyrene-b-poly(acrylic acid) vesicles and
how this enhances the photoreduction performance of the
bioconjugate.
Results and discussion
Synthesis of complexes
All asymmetric Ru(II)/Ir(III) complexes were synthesised using a
directed strategy in a two-step procedure to prevent “scram-
bling” of the terpyridine ligands. Complexes Ru(II) 1 and Ir(III)
1 were synthesised by heating the ruthenium(II) or iridium(III)
chloride salt with 2,2′:6′,2′′-terpyridine in absolute ethanol or
neat ethylene glycol, respectively, following literature methods
to yield a mono-complex.17,18 Subsequently, the second aniline
terpyridine 2 ligand was heated with the mono-complexes
Ru(II) 1 and Ir(III) 1 in ethylene glycol based on the method
adapted from Collin et al. as shown in Scheme 1 to yield Ru(II)
3 and Ir(III) 3.17
Generally, the Ru(II) mono-complex is reacted with the
second ligand in ethanol (reductant) containing N-ethylmor-
pholine (catalyst)19 and refluxed to afford the bis(terpyridine)
complex. Interestingly, it was found that in the presence of
N-ethylmorpholine, complexation with aniline terpyridine 2
resulted in N-alkylation of the ligand. As a result of the poor
yield due to the removal of catalyst in the complexation re-
action of aniline 2, the reaction was repeated using an alter-
native method based on Ir(III) bis(terpyridine) literature, using
ethylene glycol as a solvent substitute20 increasing the yield of
Ru(II) 3 from 28% to 76%.
Scheme 1 Synthesis of maleimide functionalised Ru(II)/Ir(III)-bisterpyridines.
(a) Ethylene glycol, 110 °C, Ru(II) 3, 76%; Ir(III) 3, 74%. (b) 6-Maleimidocaproic acid,
O-(7-azabenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
(HATU), N,N-diisopropylethylamine, DMF, r.t. Ru(II) 4, 41%; Ir(III) 4, 9.5%.
The complexes were purified using column chromatography
on silica or alumina and characterised by high resolution ESI
mass spectrometry. The most abundant molecular ion in mass
spectrometry corresponded to complete loss of counter ions
(PF6−), with the exception of Ru(II) 4, resulting in a loss of two
−
PF6 anions. Additionally, complexation was confirmed by
UV-Vis spectroscopy showing the appearance of the character-
1
istic metal-ligand charge transfer (MLCT) band. H NMR spec-
troscopy confirmed complexation due to the characteristic
upfield shift of the 6,6′′-protons as a result of anti to syn con-
formation change of the terpyridine ligand. The Ru(II) 3 exhi-
bited a typical shift, with a doublet observed at δ 8.73 ppm for
ligand 2 shifted to δ 7.44 ppm for Ru(II) 3.
Maleimide functionalised complexes Ru(II) 4 and Ir(III) 4
were prepared for bioconjugation to cytochrome c via Michael
addition. The maleimide functionality was introduced using
conditions adapted from Hovinen21 due to the unreactive
nature of the aniline derivatives Ru(II) 3 and Ir(III) 3 preventing
the use of classical N-hydroxysuccinimide/N,N′-dicyclohexyl-
carbodiimide (NHS/DCC) coupling conditions. Maleimide
X-ray structures of 1 and 2
functionalised Ru(II) 4 and Ir(III) 4 were prepared using the Red blades of Ru(II) 3 and Ru(II) 4 were crystallised by diffusion
peptide coupling agent O-(7-azabenzotriazole-1-yl)-1,1,3,3-tetra- of diethyl ether vapour into N,N′-dimethylformamide solutions
methyluronium hexafluorophosphate (HATU) with N,N-diisopro- of the complexes. Ru(II) 3 was crystallised in the triclinic space
ˉ
pylethylamine (DIPEA).
group (P1). As shown in Fig. 1, crystal Ru(II) 3 displays an
This journal is © The Royal Society of Chemistry 2013
Org. Biomol. Chem., 2013, 11, 4602–4612 | 4603

View Article Online
Paper
Organic & Biomolecular Chemistry
Fig. 1 ORTEP representation of the X-ray crystal structure of asymmetric Ru(II)
−
3 [Ru(C15H11N3)(C21H16N4)](PF6)2·2C3H7NO. Hydrogen atoms, PF6 anions and
solvent molecules omitted for clarity and thermal ellipsoids are plotted as 20%
probability.
Scheme 2 Synthesis of Ru(II)/Ir(III)–cyt c bioconjugates. (a) Reduced iso-1 cyto-
chrome c (10 μM), phosphate buffer (20 mM), ethylenediaminetetraacetic acid
(20 mM), tris(2-carboxyethyl)-phosphine (TCEP, 5 μM), pH 7.0, acetonitrile (5%
v/v), r.t. Ru(II)–cyt c, 27%; Ir(III)–cyt c, 33%.
orthorhombic distortion from octahedral symmetry (N2A–Ru–
N3A 78.8(5)°) which is as expected and observed in [Ru(tpy)2]-
(PF6)2.22
As shown in Table S1,† the Ru(II) 3 shows a significant twist
with a torsional angle of 29.1(2)° (C7B–C8B–C16B–C17B) about
the interannular bond, greater than the corresponding twist
found in the free 4′-phenyl terpyridine ligand.23 Ru–N bond
lengths between ligands A and B are equivalent, 2.033(7) Å and
is within reported ranges for [Ru(tpy)2](PF6)2.22 The C–C and
C–N bond lengths within the aromatic rings are normal and
average 1.380(1) and 1.355(6) Å, respectively. The interannular
C–C bond distances of ligands A and B average 1.464(5) and
1.474(7) Å and are consistent with reported asymmetric
complex [Ru(tpy)(4′-5-carboxypentyl-tpy)](PF6)2 of 1.471(1) Å.17
Maleimide functionalised complex Ru(II) 4 was crystallised
in the monoclinic space group (P21/c) with structural character-
istics similar to that of Ru(II) 3. The main notable difference is
the reduction in the torsion angle between 4′-aryl maleimide
group and the central pyridyl ring of ligand B with an angle of
23.9(8)° as shown in Table S1† and Fig. 2.
Scheme 2 are desirable due to the presence of a single avail-
able cysteine residue at CYS102 for modification with malei-
mides allowing site-specific modification. The synthesis of
bioconjugates followed previously optimised conditions in our
group.7 The bioconjugate Ru(II)–cyt c was site-specifically
reacted by using the maleimide functionalised Ru(II) 4.
Iso-1 cytochrome c was added to a solution containing five-
fold excess of Ru(II) 4 in a phosphate buffer at pH 7.0 contain-
ing ethylenediaminetetraacetic acid (EDTA) and acetonitrile.
The final reaction conditions were iso-1 cytochrome c (10 μM),
Ru(II) 4 (50 μM), phosphate buffer (20 mM), EDTA (20 mM)
and acetonitrile (5% v/v) at pH 7.0 as shown in Scheme 2. The
mixture was stirred overnight at room temperature in a plastic
reaction vessel in the dark to prevent degradation of malei-
mide and photoreduction of cytochrome c. It should be noted
that the addition of EDTA improves bioconjugation yields by
removal of trace copper ions via chelation, as oxidation of
cysteine can occur.24
Synthesis of Ru(II)/Ir(III) bioconjugates
It was observed that with the hexafluorophosphate salt of
Ru(II) 4, significant precipitation of the complex was observed
after stirring for 20 h resulting in extremely low yields <1%.
However, it was noticed that after exchange with a chloride
salt, improved water solubility of the complex Ru(II) 4 was
observed with slight formation of precipitates. Subsequently,
purification of conjugate Ru(II)–cyt c was achieved using
similar conditions as previously developed in our group for
Ru(II)-bis(terpyridine) based bioconjugates.6 Separation of co-
valently attached Ru(II)–cyt c from unreacted proteins and
ligands can be achieved using Ni2+ immobilised IMAC chro-
matography, although the mechanism remains unknown.
In addition to increasing yields by increasing complex solu-
bility with chloride counter-ion exchange, it was discovered
that the addition of half an equivalent (to cyt c) of tris(2-car-
boxyethyl)-phosphine (TCEP) improved the bioconjugate yield
(Ru(II)–cyt c) to a maximum of 27%, an approximate 20-fold
increase. TCEP is a water soluble reducing agent and when
Light-harvesting bioconjugates Ru(II)/Ir(III)–cyt c were prepared
for photoinduced electron transfer studies. Bioconjugation
reactions involving the iso-1 form of cyt c, as shown in
Fig. 2 X-ray crystal structure of the asymmetric Ru(II)
4 [(Ru(C15H11N3)-
−
(C31H27N5O3)](PF6)2·2C3H7NO·H2O. Hydrogen atoms, PF6 anions and solvent
molecules omitted for clarity and thermal ellipsoids are plotted as 20%
probability.
4604 | Org. Biomol. Chem., 2013, 11, 4602–4612
This journal is © The Royal Society of Chemistry 2013

View Article Online
Organic & Biomolecular Chemistry
Paper
used in low concentration does not react with the maleimide covalent interactions between cyt c and Ir(III)-bis(terpyridine)
functional group, unlike commonly used reducing agents such complexes which is discussed further in the next section.
as dithiothreitol (DTT) and 2-mercaptoethanol. However, when
As shown in Fig. 3, gel electrophoresis of Ir(III)–cyt c shows
used at an equivalent concentration to Ru(II) 4, the reaction is that the bioconjugate (lane 3) migrates as a slightly larger
inhibited due to the competing reaction of Ru(II) 4 with TCEP. species than unmodified iso-1 cyt c (lane 1), which is as
This is in agreement with literature, as Schafer et al.25 found expected. Also, the presence of unreacted cyt c in Ir(III)–cyt c
that at pH 7, 50% of N-ethylmaleimide reacted with an equi- was also confirmed by gel electrophoresis, as a faint band can
molar amount of TCEP in 5 minutes. Hence, the cysteine be discerned below the main band in lane 3. A faint band
residue (CYS102) of cyt c can oxidise to form sulfoxide species between 3 and 6 kDa was observed in lane 3. The identity of
resulting in low yields. The presence of TCEP in the reaction this band is not known. An in-gel tryptic digest of each band
mixture ensured that the CYS102 residue remained reduced in this gel was inconclusive, only confirming that cyt c was
and available for reactions with the maleimide functionalised present in all bioconjugate bands. The smaller molecular
complex Ru(II) 4 and Ir(III) 4.
weight band may be a result of fragmentation of cyt c during
The bioconjugates were characterised using UV-Vis spectro- bioconjugation and isolation.
scopy, MALDI-TOF mass spectrometry and gel electrophoresis
(SDS-PAGE). Gel electrophoresis of Ru(II)–cyt c, as shown in
Non-covalent binding between Ir(III) bis(terpyridine) and cyt c
Fig. 3 shows that the bioconjugate (lane 2) migrates as a
slightly larger species than unmodified iso-1 cyt c (lane 1),
which is as expected.
Ir(III)-bis(terpyridine) complexes are capable of room tempera-
ture luminescence under aerated conditions. As such, Stern–
Volmer analysis can be used to identify interactions between a
fluorescent or phosphorescent probe and a quencher.26,27 To
investigate possible non-covalent interactions between Ir(III)
bisterpyridine complexes and iso-1 cyt c, the effect of cyt c con-
centration on the emission of a reference Ir(III) complex, Ir(III)
6 was examined as shown in Fig. 4.
Equimolar solutions of Ir(III) 6 with varying concentrations
of cyt c were prepared and the emission intensity was recorded
at 350 nm. The Stern–Volmer plot of emission intensity of
Ir(III) 6 (4.3 μM) at 25 °C and 35 °C as a function of cyt c con-
centration is shown in Fig. 5.
Based on a simple 1 : 1 stoichiometric model and complete
quenching upon complexation, the data corresponds to
binding isotherms and can be analysed by non-linear
regression to give an association constant (Ka) for the Ir(III)
6 : cyt c interaction.28 This analysis gave association constants
of (3.5 0.6) × 104 M−1 at 25 °C and (3.3 0.9) × 104 M−1 at
35 °C showing temperature independence on the binding of
Ir(III) 6 to cyt c. The Stern–Volmer plot as shown in Fig. 5 is
non-linear and displays an upward curvature concave towards
the axis of ordinates and second order with respect to cyt c
concentration. This is typical of a combination of static and
dynamic binding.27
The Ir(III) 4 with the hexafluorophosphate anion was
exchanged with chloride, which improved water solubility to
such an extent that no acetonitrile was required to solubilise it
in bioconjugation reactions. Bioconjugation was carried out
under similar conditions to Ru(II)–cyt c, with 0.5 equivalent of
TCEP (to cyt c). Similarly, purification of the reaction mixture
followed the purification protocol of Ru(II)–cyt c. Characteris-
ation of the Ir(III)–cyt c bioconjugate by UV-Vis, MALDI-TOF
and gel electrophoresis showed incomplete removal of
unreacted cyt c and Ir(III) 4. Based on UV-Vis spectra, it is esti-
mated that the purified fractions contained 20% of unreacted
Ir(III) 4 (Fig. S5, ESI†). This is attributed to the strong non-
Moreover, to further investigate the photoreduction
rate previously observed for non-covalent mixtures of Ir(III) 6
and iso-1 cyt
c in the presence of sacrificial donor
Fig. 3 Gel electrophoresis (reduced) of SeeBlue® Plus2 molecular weight
marker (lane 1), iso-1 cytochrome c (lane 2, expected 12 706), Ru(II)–cyt c (lane
3, expected 13 559) and Ir(III)–cyt c (lane 4, expected 13 649). Samples are
reduced with dithiothreitol (DTT). Samples stained with SimplyBlue™ safestain.
Fig. 4 Ru(II) and Ir(III) reference compounds used in this work.
This journal is © The Royal Society of Chemistry 2013
Org. Biomol. Chem., 2013, 11, 4602–4612 | 4605

View Article Online
Paper
Organic & Biomolecular Chemistry
Fig. 5 Stern–Volmer plot of Ir(III) 6 (4.3 μM) emission intensity (at 25 °C and
35 °C) in the presence of cyt c.
Fig. 7 Room temperature photoreduction of Ru(II)/Ir(III)-bis(terpyridine) based
cyt c samples. All sample measurements were made at a concentration of 2.3
0.1 μM in 5 mM sodium dihydrogen phosphate, 5 mM ethylenediaminetetra-
■
acetic acid (EDTA), pH 7.0. Ru(II)–cyt c ( ), Ru(II)–cyt c:polymersome ( ), Ru(II)–cyt
c non-degassed ( ), Ru(II)–cyt c no EDTA ( ), non–covalent (1 : 1) Ru(II) 5 : cyt c
mixture ( ), non-covalent (1 : 1) Ru(II) 5 : cyt c:polymersome mixture ( ) and
Ir(III)–cyt c ( ). Error bars indicate standard deviation. Ru(II)/Ir(III)-bis(terpyridine)
complexes irradiated with 465 nm or 372 nm light, respectively.
cytochrome c bioconjugates using a short and long chain
spacer.6 It was found that long chain spacers resulted in
optimum electron transfer as the use of short chain spacers
caused inactivation of the protein.
Based on the findings of Peterson et al.,6 photoreduction
studies of the novel long chain spacer bioconjugate Ru(II)–cyt c
and non-covalent mixtures with reference Ru(II) 5 and iso-1
cyt c as shown in Fig. 4 and Scheme 2 were used to
determine the effect of membrane encapsulation on photo-
reduction rates. Additionally, photoreduction studies were per-
formed on bulk solution Ir(III)–cyt c to determine the effect of
Fig. 6 The effect of Ir(III) 6 on the initial rate of cyt c heme reduction in non-
covalent mixtures of Ir(III) 6, iso-1 cyt c (8 μM), EDTA (20 mM), phosphate buffer
(20 mM), pH 7.0, 25 °C.
substituting
(Fig. 7).12,29
a long-lived luminescence lifetime complex
ethylenediaminetetraacetic acid (EDTA), the effect of Ir(III) con-
centration on the rate of cyt c heme reduction was studied as
shown in Fig. 6.
As shown in Fig. 6, the initial rate of cytochrome c
reduction is only dependent on the concentration of the
Ir(III) bis(terpyridine) complex at low Ir(III) concentration
(below 4 μM). No further increase in the initial rate of
reduction is observed at concentrations greater than a ∼1 : 1
ratio of Ir(III) to iso-1 cyt c, consistent with the applied 1 : 1
In order to demonstrate light-activated electron transfer in
bioconjugate Ru(II)–cyt c, samples were prepared in a special-
ised small volume cuvette, degassed and irradiated (2.5 cm)
with a constant area at a bioconjugate concentration of 2.3
0.1 μM and an equivalent 1 : 1 non-covalent mixture of Ru(II) 5
and iso-1 cyt c in a 5 mM phosphate buffer, 5 mM EDTA at pH
7.0 in either bulk solution or encapsulated in the PS140-b-
PAA48 membrane. This experiment was also conducted under
different conditions to determine the effect on heme reduction
rate, including the presence of oxygen, the absence of sacrifi-
cial electron donor EDTA or the substitution of Ru(II) 4 with
Ir(III) 4 of the corresponding bioconjugate.‡ It should be noted
that concentrations used for photoreduction studies were low
binding constant model. The rapid reduction of cyt
c
under these conditions is believed to be due to both the
non-covalent binding mechanism between the Ir(III) complex
and iso-1 cyt c, as well as a secondary reduction mechanism by
EDTA radicals.
Photoreduction studies of Ru(II)/Ir(III) bioconjugates
‡The concentrations were estimated by UV-Vis absorption spectroscopy with
molar absorption coefficients: iso-1 cyt c/Ru(II)–cyt c/Ir(III)–cyt c (ε410 = 97.6 mM−1
Our group has previously demonstrated room temperature
photoinduced electron transfer in Ru(II)-bis(terpyridine) cm−1 39 and Ru(II) 5 (ε476 = 17.7 mM−1 cm−1).8
)
4606 | Org. Biomol. Chem., 2013, 11, 4602–4612
This journal is © The Royal Society of Chemistry 2013

View Article Online
Organic & Biomolecular Chemistry
Paper
Table 1 Estimated rates of heme reduction, reduction efficiency and quantum
efficiency (Φ) for the photoinduced reduction of Ru(II)–cyt c/Ir(III)–cyt c systems
Φd (%)
Heme reduction Efficiencyi,c (%) (electrons/
(electrons per
(electrons/
photons)
absorbed
photons)
Samplea
λ (nm) second)b
Ru(II)–cyt ce
465
7.9 1.8 × 109 5.6 0.9 × 10−5 5.9 1.5 × 10−4
1.5 0.3 × 1010 1.1 0.3 × 10−4 1.1 0.3 × 10−3
4.6 0.6 × 109 3.3 0.6 × 10−5 3.4 0.6 × 10−4
5.1 1.3 × 109 3.6 1.0 × 10−5 3.8 1.1 × 10−4
6.6 0.4 × 1011 3.0 1.5 × 10−2 3.8 1.9 × 10−1
Ru(II)–cyt cf 465
Ir(III)6 : cyt ce,g 465
Ir(III)6 : cyt cf,g 465
Ir(III)–cyt ce,h 372
a Sample concentration of 2.3 0.1 μM in 5 mM sodium dihydrogen
phosphate, 5 mM EDTA, pH 7.0 at 25 °C with 80 μL volume. Irradiated
with 20 2.3 mW cm−2 of 465 nm light or 0.04 0.02 mW cm−2 of
372 nm light. b Initial rate of heme reduction was estimated using
amount of protein reduced in the first 1860 s interval. c Efficiency (%)
was determined by dividing initial rate of heme reduction by incident
photons. d Quantum efficiency was determined by correcting for the
optical density of the solutions (Fig. S2, ESI). e Bulk solution
measurement. f Encapsulated measurement in PS140-b-PAA48
membrane. g Non-covalent mixture. h 120 μL volume. Errors are
standard deviation. i Incident photons are 1.40 0.2 × 1016 photons
s−1 for 465 nm light or 2.2 1.1 × 1013 photons s−1 for 372 nm light.
Incident photons calculated from power output over an irradiation
area of 1.0 × 0.3 cm for the light source.
Fig. 8 TEM micrograph of polystyrene140-b-poly(acrylic acid)48 (PS140-b-PAA48
)
polymersome aggregates in the presence of 14 μM Ru(II)–cyt c in PBS (25 °C).
Average polymersome diameter of 290 132 nm. Scale bar: 200 nm.
to prevent intermolecular electron transfer in bioconjugate
samples.
corresponding to a ∼650-fold increase in quantum efficiency
relative to bulk Ru(II)–cyt c. This significant improvement may
be due to the longer luminescence lifetime of Ir(III)-bis(terpyri-
dine) complexes at room temperature.12,29
We have previously reported that positively charged pro-
teins and peptides, including cyt c, are capable of inducing
polymersome formation using the diblock copolymer PS140
-
b-PAA48 with concomitant encapsulation in the membrane.30
To determine the effect of electron transfer after encapsulation
within PS140-b-PAA48 polymersome membranes, the polymer
aggregates were characterised by transmission electron
microscopy (TEM) prior to room temperature photoreduction
measurements to ensure that bioconjugate Ru(II)–cyt c induced
polymersome formation. As shown in Fig. 8, polymersome
aggregates were observed with an average diameter of 290
132 nm.
To determine the effect of covalent linkage of Ru(II)-bis(ter-
pyridine) donor to cyt c, non-covalent studies using
a
1 : 1 mixture of reference complex Ru(II) 5 and cyt c in bulk
solution or encapsulated in membrane were performed in the
presence of EDTA and degassed. Ru(II) 5 was chosen as a
control complex compared to Ru(II) 4 as the lack of maleimide
functionality prevents reaction with protein. It was observed in
Fig. 7 that a dramatic decrease in photoreduction yield
resulted in non-covalent mixtures. This indicates that covalent
attachment to protein is essential to ensure proximity between
donor and acceptor to increase photoreduction efficiency.
However, the behaviour of increasing Φ was observed after
encapsulation, consistent with covalent bioconjugate Ru(II)–cyt
c studies. The reduction efficiencies and quantum efficiencies
of bioconjugate Ru(II)–cyt c and non-covalent mixtures in bulk
solution and membrane encapsulation is summarised in
Table 1.
Photoexcitation of Ru(II)-bis(terpyridine) to the triplet
metal-to-ligand charge-transfer (3MLCT) state is short
lived and non-luminescent at room temperature with
an excited-state lifetime estimated to be 250 ps.35 To investi-
gate if the presence of oxygen significantly quenches
the excited chromophore Ru(II)*, photoreduction studies
were performed without degassing. A quenching of photo-
reduction by ≈50% was observed in non-degassed experiments
as shown in Fig. 7. Additionally, the presence of sacrificial
electron donor, EDTA, is essential as bioconjugate Ru(II)–cyt c
in phosphate buffer shows negligible photoreduction as
shown in Fig. 7.
As shown in Fig. 7, encapsulation of Ru(II)–cyt c resulted in
an increase in initial rate of reduction of heme and fully
reduced within 50 minutes. Encapsulation was estimated to be
double the Φ of bulk solution Ru(II)–cyt c photoreduction with
a Φ of 1.1 0.3 × 10−3% over an estimated maximum distance
between ruthenium and heme centre of ≤32 Å (Fig. S3, ESI†).
The increase in Φ could be explained using semi-classical
theory (Marcus–Hush theory of electron transfer) which
describes electron tunnelling in proteins.31–33 It has
been reported that embedding reactants in a membrane
(low dielectric medium) dramatically reduces reorganisational
energies, hence, the encapsulation of Ru(II)–cyt c in the
polymersome membrane leads to an increase in electron
transfer rate (kET).32 Another possibility leading to increased
Φ may be due to the flexible linker used in Ru(II) 4 for attach-
ment to cyt c allowing the complex to lie flat on the protein
surface when embedded in the polyelectrolyte membrane. This
decrease in distance between the donor and acceptor resulted
34
in increased kET
.
As shown in Table 1, it was observed that Ir(III)–cyt c had a
of 3.8 1.9 in the bulk solution state,
10−1
Φ
×
%
This journal is © The Royal Society of Chemistry 2013
Org. Biomol. Chem., 2013, 11, 4602–4612 | 4607

View Article Online
Paper
Organic & Biomolecular Chemistry
dimethylformamide (DMF) was purchased from Sigma Aldrich
and used directly from the bottle. Deuterated solvents for NMR
Conclusions
Ruthenium(II) and iridium(III) bis(terpyridine) complexes were were obtained from Cambridge Isotope Laboratories. For
prepared with maleimide functionalities in order to site- aggregation studies and preparation of all salt buffers, ultra
specifically modify yeast iso-1 cytochrome c possessing a pure water (R > 18 × 106 Ω) was used. Water and organic
single cysteine residue available for modification (CYS102). solvent was filtered through a 0.45 μm cellulose membrane
Single X-ray crystal structures were solved for aniline and mal- filter (Minisart RC 25, Sartonius Stedim Biotech) prior to
eimide complexes Ru(II) 3 and Ru(II) 4, respectively, providing polymer aggregation studies. Diblock copolymer, poly-
detailed structural detail of the complexes. Light-activated bio- styrene140-b-poly(acrylic acid)48 (PDI = 1.10), was purchased
conjugates prepared from Ru(II) 4 and Ir(III) 4 were prepared from Encapson (The Netherlands, catalogue number 1036). All
allowing for room temperature photoinduced electron transfer other chemicals were used as received.
studies. It was observed that strong binding between Ir(III) bis
Yeast cytochrome c from Saccharomyces cerevisiae (catalogue
(terpyridine) complexes and cyt c occurred, confirmed via titra- number C2436) was purchased from Sigma Aldrich. Yeast iso-1
tion and Stern–Volmer studies with Ir(III) 6. As a result of the cytochrome c was purified following previously published pro-
high binding affinities between Ir(III) bis(terpyridine) com- cedures prior to bioconjugation reactions.7,39 Buffer pH values
plexes and cyt c, difficulties in isolation of Ir(III)–cyt c were were monitored using a Scholar 425 pH meter (Corning) and
encountered resulting in ∼20% of Ir(III) 4 present in the Ir(III)– filtered using a 0.45 μm (Millipore, 47 mm regenerated cellu-
cyt c bioconjugate mixture.
lose) prior to use.
Photoinduced electron transfer studies of Ru(II)–cyt c in
A Varian Cary 50 Bio UV-Vis or Cary 5 UV-Vis-NIR spectro-
bulk solution and polymer membrane encapsulated specimens meter was used for UV-Vis spectra measurements. Fluo-
were performed using EDTA as a sacrificial electron donor. It rescence spectra were recorded using a Varian Cary Eclipse
was found that membrane encapsulation of Ru(II)–cyt c in spectrometer with excitation and emission slits at 5 nm, exci-
PS140-b-PAA48 resulted in a two-fold increase of the quantum tation filter “auto”, emission filter “open” and PMT voltage set
efficiencies. Moreover, Ir(III)–cyt c bioconjugates showed a to “medium”, unless otherwise stated. NMR spectra (1H and
∼640-fold increase in quantum efficiencies relative to Ru(II)– 13C) were recorded in the designated solvents using a Bruker
cyt c. The bioconjugates allow photo-switchability between Avance DPX (300 MHz) spectrophotometer. Chemical shifts are
on/off states controlling the reduction of cyt c. The biohybrids measured in parts per million (ppm), internally referenced
potentially will allow scientists to drive biological processes via relative to tetramethylsilane (SiMe4, 1H and 13C = 0 ppm) or
room temperature photoinduced electron transfer. Potential residual solvent peaks (CD3CN: 1H = 1.94 and 13C = 1.32;
1
1
applications include the encapsulation in artificial capsules DMSO-d6: H = 2.50, 13C = 39.52; CDCl3: H = 7.26 ppm, 13C =
and vesicles as a component of an artificial electron transport 77.16 ppm). IR spectra were recorded on Shimadzu
a
chain to drive chemical reactions or energy gradients as nano- FTIR-8400S, ThermoNicolet Avatar model 370 FT-IR or a
reactors or artificial cells.36–38
Perkin Elmer Spotlight 400 FT-IR spectrometer equipped with
a microscope and attenuated total reflectance (ATR) acces-
sories with diamond crystal inset. Low resolution electrospray
ionisation (ESI) mass spectra were recorded on a Waters Micro-
mass ZQ electrospray instrument. High resolution ESI mass
spectrometry was performed on a Thermo Linear Quadropole
Experimental
Chemicals, solvents and materials
Chemicals were purchased from Sigma Aldrich with the excep- Ion Trap Fourier Transform Ion Cyclotron Resonance (LQT FT
tions of ammonium acetate, sodium dihydrogen phosphate, Ultra) mass spectrometer in electrospray mode with a 7 T
sodium hydroxide, sodium bicarbonate, anhydrous sodium superconducting magnet. MALDI-TOF mass spectra were
sulphate, citric acid, iodine, pyridine and hydrazine mono- recorded on an Applied Biosystems Voyager DE STR MALDI
hydrate were purchased from Ajax Finechem Pty. Ltd, 4-nitro- reflectron TOFMS (protein and bioconjugate measurements
benzaldehyde (Hopkins and Williams Ltd), ammonium were made in linear mode). Melting points were recorded on a
hexafluorophosphate (Acros Organics) and ruthenium(III) tri- Mel-temp II hot stage apparatus. TEM micrographs were
chloride hydrate (Precious Metals Online). Silica was pur- recorded on a JEOL 1400 (80 kV) instrument.
chased from Davisil (40–63 μM). Thin layer chromatography
Protein and bioconjugate purification was performed using
plates (Kieselgel 60 F-254 pre-coated sheets 0.25 mm) were pur- a GE Healthcare ÄKTApurifier. Cation exchange chromato-
chased from Merck. Neutral alumina oxide was purchased graphy (CEX) was performed using a strong cation exchange
from Merck (Alumina Oxide 90 active neutral, 20–230 mesh). column (TSKgel SP-5PW, Supelco). Immobilised metal affinity
Dichloromethane (CH2Cl2) and methanol (CH3OH) were dis- chromatography (IMAC) was performed using either a Ni2+
tilled before use. Dry solvents such as acetonitrile (CH3CN), charged HisTrap HP (GE Healthcare, 1 mL) or an Acrosep
dichloromethane, diethyl ether (Et2O) and tetrahydrofuran Hypercel (Pall, 1 mL) column. Protein solutions were concen-
(THF) were obtained from a Pure Solv dry solvent system (Inno- trated using 3000 molecular weight cut-off (MWCO) centrifuge
vative Technology, Inc., model #PS-MD-7). Dry methanol was concentrators (Amicon Ultra-15, Amicon Ultra-4 or Amicon
distilled and stored over calcium chloride. Dry N,N- Ultra-0.5, Millipore). Samples were dialysed into MilliQ water
4608 | Org. Biomol. Chem., 2013, 11, 4602–4612
This journal is © The Royal Society of Chemistry 2013

View Article Online
Organic & Biomolecular Chemistry
Paper
using Slide-A-Lyzer Mini Dialysis Units (3500 MWCO, Pierce) integration, reduction with multi-scan absorption correction
or Tube-O-Dialyzer Dialysis Units (4000/50 000 MWCO, G method was carried out using Bruker APEX2 Suite software.40
Biosciences).
The structure was solved by Direct Methods program
Ru(II)/Ir(III)–cyt c bioconjugate photo-reactions were per- SHELXS-97 and refined by full-matrix least-squares refinement
formed using a 16 LED Blue (465 nm) Flashlight (LDP LLC) or program SHELXL.41 All non-hydrogen atoms were refined ani-
a 16 LED White (372 nm) Flashlight (LDP LLC), respectively. sotropically and hydrogen atoms were included by using a
Power measurements of LED Flashlights were made using a riding model. Further information is available in the ESI CIF
Newport Power Meter (Model 1918-C).
files.†
X-ray structure determination of Ru(II) 4. The X-ray diffrac-
tion measurement for Ru(II) 4 was carried out at MX1 beamline
Gel electrophoresis
Gel electrophoresis was performed using Invitrogen Novex® at the Australian Synchrotron Facility, Melbourne. The crystal
NuPage® 12% Bis-Tris, mm, 10-well gels, SeeBlue® was mounted on the goniometer using cryo loop for intensity
1
Plus2 molecular weight marker, NuPage® LDS sample buffer measurements, coated with paraffin oil and immediately trans-
(4×), NuPage® sample reducing agent (10×), NuPage® MES ferred to the cold stream using a Cryostream attachment. Data
SDS running buffer, SimplyBlue™ safestain and the gels run was collected using Si<111> monochromated synchrotron
using a Zoom Dual Power supply (model ZP10002, Invitrogen). X-ray radiation (λ = 0.71023 Å) at 100(2) K and was corrected
Samples for gel electrophoresis were prepared by dilution in for Lorentz and polarization effects using the XDS software.42
Novex® NuPage® LDS sample buffer (Invitrogen). Samples The structure was solved by Direct methods and the full-matrix
were reduced (to eliminate disulfide dimers) by adding least-squares refinements was carried out using SHELXL.41
NuPage® sample reducing agent (active ingredient dithiothrei- Further information is available in the ESI CIF files.†
tol (DTT)). Samples were heated at 70 °C for 10 min to
[Ru(tpy)(4′-(4-aminophenyl)-2,2′:6′,2′′-tpy)](PF6)2 (Ru(II) 3)
denature the protein. Novex® NuPage® gels (12% Bis-Tris,
10-wells) were then loaded with 1–3 μg of protein per well, run
Method 1. A solution of 4′-(4-aminophenyl)-2,2′:6′,2′′-terpyri-
at a constant 200 V for 40 min and stained according to the dine 2 (273.3 mg, 0.8425 mmol) and [Ru(tpy)]Cl3 1 (373.6 mg,
procedure included with SimplyBlue™ safestain.
0.8447 mmol) in ethylene glycol (50 mL) was heated to 110 °C
for 20 h under nitrogen. Reaction mixture was diluted to
150 mL with water and filtered over celite. Filtrate was precipi-
Transmission electron microscopy (TEM) studies
TEM samples were prepared by placing 20 μL of sample onto a tated using ammonium hexafluorophosphate, washed with
formvar-coated copper grid and the excess water was blotted water (3 × 50 mL) and collected with acetonitrile. Purified over
away after 2 min with a filter paper. For statistical analysis, a silica using a gradient from 90 : 9 : 1 CH3CN : H2O : KNO3 (satu-
population of 100 polymersomes were measured from TEM rated) to 20 : 3 : 1 CH3CN : H2O : KNO3 (saturated). Fractions
micrographs for determination of the average and standard precipitated with ammonium hexafluorophosphate and
deviation of diameters.
washed with water. Product further purified over alumina
(neutral) using a gradient from acetonitrile to 90 : 9 : 1 CH3CN :
H2O : KNO3 (saturated). Fractions pooled, precipitated with
X-ray crystallography
Crystal growth of Ru(II)-bis(terpyridine) complexes. In ammonium hexafluorophosphate, washed with water (3 ×
general, Ru(II)-bis(terpyridine) complex (ca. 5 mg) was dis- 20 mL) and collected with acetonitrile yielding Ru(II) 3 as a red
solved in minimal solvent (DMF) resulting in a concentrated solid (609.6 mg, 76%). Mp >274 °C (decomposed); 1H NMR
solution. Crystals were grown by slow diffusion of anhydrous (300 MHz, CD3CN) δ 8.90 (s, 2H), 8.72 (d, J = 7.9 Hz, 2H), 8.60
diethyl ether into a solution of complex in N,N-dimethylform- (d, J = 7.9 Hz, 2H), 8.48 (d, J = 7.9 Hz, 2H), 8.38 (t, J = 8.3 Hz,
amide at room temperature or 4 °C for Ru(II) 3 and Ru(II) 4, 1H), 8.00 (dt, J = 8.6, 2.4 Hz, 2H), 7.91 (tt, J = 7.9, 1.3 Hz, 4H),
respectively. A suitable single crystal was selected under a 7.44 (dd, J = 4.9, 0.8 Hz, 2H), 7.30 (dd, J = 4.5, 0.8 Hz, 2H),
polarising microscope (Leica M165Z) for single crystal X-ray 7.21–7.09 (m, 4H), 6.95 (dt, J = 8.7, 2.4 Hz, 2H); 13C NMR
diffraction analysis.
(75 MHz, CD3CN) δ 159.1, 156.5, 156.0, 153.5, 153.3, 144.9,
X-ray structure determination of Ru(II) 3. The X-ray diffrac- 138.9, 136.4, 129.8, 128.4, 128.2, 125.3, 124.6, 120.9, 115.8;
tion measurement for Ru(II) 3 was performed on a Bruker IR (ATR) νmax/cm−1 3646 (w), 3593 (w), 3506 (w), 3414 (w),
kappa APEX-II CCD diffractometer at 150 K by using graphite- 3121 (w), 1990 (w), 1633 (m), 1597 (m), 1529 (w), 1430 (m);
monochromated Mo-Kα radiation (λ = 0.71075 Å). The crystal UV-Vis (CH3CN) λmax/nm (ε/M−1 cm−1) 490 (1.71 × 104), 364
was mounted on the goniometer using cryo loops for intensity (9.80 × 103), 308 (5.00 × 104), 283 (2.55 × 104), 272 (2.86 × 104),
measurements, coated with paraffin oil and immediately trans- 231 (2.67 × 104); HRMS (ESI) m/z: ([M − PF6)]+) calcd for
ferred to the cold stream using Oxford Cryostream 700 system C36H27N7P1F6Ru+, 804.1023; found, 804.1006. MS (ESI) m/z:
attachment. Upon obtaining an initial refinement of unit cell ([M − 2PF6]2+) calcd for C36H27N7Ru2+, 329.57; found, 329.49.
parameters, the data collection strategy was calculated to
Method 2. A solution of 4′-(4-aminophenyl)-2,2′:6′,2′′-terpyri-
achieve a redundancy of at least 4 throughout the resolution dine 2 (179.0 mg, 0.5518 mmol) and [Ru(tpy)]Cl3 1 (248.8 mg,
range (∞–0.80 Å) at 10 s exposure time per frame utilising the 0.5645 mmol) in ethanol (100 mL) was refluxed for 20 h under
kappa offsets on the four circle goniometer geometry. Data nitrogen. Reaction mixture was filtered over celite, concentrated
This journal is © The Royal Society of Chemistry 2013
Org. Biomol. Chem., 2013, 11, 4602–4612 | 4609

View Article Online
Paper
Organic & Biomolecular Chemistry
in vacuo and diluted with water (100 mL). Filtrate was precipi- degassed with three freeze–thaw cycles, heated to 160 °C
tated using ammonium hexafluorophosphate, washed with under nitrogen in the dark, and stirred at this temperature for
water (3 × 50 mL) and collected with acetonitrile. The product 15 minutes. The mixture was left to cool before the addition of
was recrystallised with acetonitrile/diethyl ether yielding Ru(II) aqueous ammonium hexafluorophosphate (90 mL). The result-
3 as a red solid (138.7 mg, 28%). Characterisation data was ing precipitate was centrifuged, washed with aqueous
identical to the compound obtained from method 1 above.
ammonium hexafluorophosphate (10 mL), absolute ethanol
(3 × 10 mL), diethyl ether (3 × 10 mL), and recrystallised twice
from acetonitrile and diethyl ether to yield Ir(III) 3 (272 mg,
74%) as an orange solid. Mp >300 °C; 1H NMR (300 MHz,
[Ru(tpy)(maleimide-hexylcarboxamido-phenyl-tpy)](PF6)2
(Ru(II) 4)
A solution of 6-maleimidocaproic acid (53.2 mg, 0.252 mmol), CD3CN) δ 8.93 (s, 2H), 8.87–8.71 (m, 3H), 8.67 (ddd, J = 8.2,
O-(7-azabenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexa- 1.4, 0.7 Hz, 2H), 8.57 (ddd, J = 8.1, 1.4, 0.7 Hz, 2H), 8.26–8.14
fluorophosphate (HATU, 95.9 mg, 0.252 mmol) and N,N-diiso- (m, 4H), 8.06 (d, J = 8.8 Hz, 2H), 7.69 (ddd, J = 5.7, 1.4, 0.6 Hz,
propylethylamine (66.8 mg, 0.517 mmol) in dry 2H), 7.54 (ddd, J = 5.7, 1.5, 0.7 Hz, 2H), 7.52–7.40 (m, 4H), 6.97
dimethylformamide (5 mL) was stirred at room temperature (d, J = 8.8 Hz, 2H), 5.06 (s, 2H); 13C NMR (75 MHz, CD3CN) δ
under nitrogen for 1 h. Subsequently, Ru(II) 3 (71.7 mg, 154.8, 154.3, 154.2, 143.8, 143.6, 131.0, 130.7, 130.4, 128.3,
0.0756 mmol) in dry dimethylformamide (5 mL) was added to 128.0, 127.5, 122.5, 115.7; IR (ATR) νmax/cm−1 3436 (br),
the mixture and stirred for a further 26 h in the dark at room 3063 (w), 2924 (w), 2857 (w), 1605 (s), 1474 (m), 1403 (m),
temperature under nitrogen. Dichloromethane (50 mL) was 1241 (m), 1103 (m), 1051 (m), 824 (s, PF6); HRMS (ESI) m/z:
added to the solution and the organic phase washed with ([M − PF6)]+) calcd for C36H27N7P2F12Ir+, 1040.1241; found,
aqueous citric acid (10% w/v, 2 × 20 mL), water (3 × 10 mL) 1040.1236. MS (ESI) m/z: ([M
−
2PF6])2+ calcd for
and dried over anhydrous sodium sulphate. Dichloromethane C36H27F6N7PIr2+, 447.58; found, 447.40.
was removed in vacuo and the concentrated red dimethylform-
amide phase containing product was precipitated from dry
[Ir(tpy)(maleimide-hexylcarboxamido-phenyl-tpy)](PF6)3 (Ir(III) 4)
diethyl ether and the solid collected by filtration, washed with A solution of 6-maleimidocaproic acid (78.5 mg, 0.372 mmol),
diethyl ether and collected with acetonitrile. Product was puri- HATU (142.2 mg, 0.372 mmol) and N,N-diisopropylethylamine
fied over silica using a gradient from acetonitrile to 70 : 29 : 1 (109.5 mg, 0.847 mmol) in dry dimethylformamide (7.5 mL)
CH3CN : H2O : KNO3 (saturated). Fractions pooled, precipitated was stirred at room temperature under nitrogen for 1 h. Ir(III) 3
with ammonium hexafluorophosphate, washed with water (3 × (129.6 mg, 0.112 mmol) in dry dimethylformamide (7.5 mL)
20 mL) and collected with acetonitrile yielding Ru(II) 4 as a red was then added to the reaction mixture, which was stirred at
solid (27.9 mg, 41%). Mp >246 °C (decomposed); 1H NMR room temperature under nitrogen in the dark for 25 h.
(300 MHz, CD3CN) δ 8.98 (s, 2H), 8.75 (d, J = 8.3 Hz, 3H), 8.63 Dichloromethane (75 mL) was added to the mixture, which
(d, J = 7.9 Hz, 2H), 8.50 (d, J = 7.9 Hz, 2H), 8.40 (t, J = 8.3 Hz, was subsequently washed with aqueous citric acid (10% w/v,
1H), 8.16 (dd, J = 8.7, 1.5 Hz, 2H), 8.00–7.85 (m, 6H), 7.43 2 × 30 mL) and water (3 × 20 mL). The aqueous phase was
(dd, J = 5.6, 0.8 Hz, 2H), 7.37 (dd, J = 6.4, 0.8 Hz, 2H), back-extracted with dichloromethane (2 × 30 mL), and the
7.20–7.10 (m, 3H), 6.75 (s, 2H), 3.49 (t, J = 7.0 Hz, 2H), 2.41 (t, organic phases were pooled, dried over anhydrous sodium sul-
J = 7.4 Hz, 2H), 1.80–1.56 (m, 5H), 1.45–1.33 (m, 2H); 13C NMR phate, and the dichloromethane removed in vacuo. The red di-
(75 MHz, CD3CN) δ 173.60, 172.51, 159.49, 159.41, 159.26, methylformamide phase containing product was precipitated
156.56, 153.61, 153.48, 149.09, 142.43, 139.19, 136.87, 135.37, into diethyl ether, and the solid was centrifuged, washed with
132.53, 129.51, 128.61, 128.56, 125.64, 125.56, 124.85, 122.17, diethyl ether (2 × 5 mL), dissolved in acetonitrile, recrystallised
121.17, 38.44, 37.76, 29.17, 27.21, 25.94; IR (ATR) νmax/cm−1 into diethyl ether and the crude solid purified via column
3647 (w), 3403 (w), 3112 (w), 2933 (w), 2857 (w), 1769 (w), chromatography (silica, 20 : 3 : 1 CH3CN : H2O : KNO3 (satu-
1699 (s), 1595 (m), 1523 (m), 1449 (m), 1407 (m); UV-Vis rated)) to yield Ir(III) 3 (11.2 mg, 9.5%) as an orange-yellow
1
(CH3CN) λmax/nm (ε/M−1 cm−1) 485 (2.60 × 104), 410 (4.88 × solid. Mp >220 °C (decomposed); H NMR (300 MHz, CD3CN)
103), 308 (7.85 × 104), 282 (4.32 × 104), 272 (4.65 × 104); HRMS δ 9.03 (s, 2H), 8.89–8.73 (m, 3H), 8.69 (ddd, J = 8.9, 1.7, 1.0 Hz,
(ESI) m/z: ([M − PF6])+ calcd for C46H38N8O3P1F6Ru+, 997.1618; 2H), 8.58 (ddd, J = 8.1, 1.4, 0.7 Hz, 2H), 8.29–8.12 (m, 6H),
found, 997.1741 and ([M − 2PF6])2+ calcd for C46H38N8O3Ru2+, 8.03–7.96 (ddd, 2H), 7.68 (ddd, J = 5.7, 1.5, 0.6 Hz, 2H), 7.57
426.0988; found, 426.1050. MS (ESI) m/z: ([M − 2PF6])2+, (ddd, J = 5.7, 1.5, 0.6 Hz, 2H), 7.53–7.42 (m, 4H), 6.75 (s, 2H),
426.11.
3.49 (t, J = 7.0 Hz, 2H), 2.42 (t, J = 7.4 Hz, 2H), 1.67 (ddt, J =
Prior to bioconjugation, Ru(II) 4 was exchanged with chloride 29.9, 14.8, 7.3 Hz, 4H), 1.46–1.27 (m, 2H); 13C NMR (75 MHz,
salt to increase solubility and yield.
CD3CN) δ 173.08, 159.00, 158.85, 155.31, 154.35, 154.23,
144.60, 143.78, 143.71, 135.16, 130.71, 130.58, 130.42, 130.23,
128.31, 128.19, 127.52, 124.37, 120.78, 38.21, 37.60, 28.93,
[Ir(tpy)(4′-(4-aminophenyl)-2,2′:6′2′′-tpy)](PF6)3 (Ir(III) 3)
[Ir(tpy)]Cl3 1 (170.0 mg, 0.320 mmol) and 4′-(4-aminophenyl)- 26.94, 25.56; IR (ATR) vmax/cm−1 3637 (w), 3390 (w), 3987 (w),
2,2′:6′,2′′-terpyridine 2 (101.3 mg, 0.312 mmol) were crushed 2925 (w), 2850 (w), 1751 (w), 1703 (s), 1591 (m), 1524 (m),
together with a glass rod in a round-bottomed flask. Ethylene 1479 (m), 1453 (m), 1412 (m), 1360 (m), 1315 (m), 1252 (m),
glycol was added (15 mL) and the reaction mixture was 1188 (m); UV-Vis (H2O) λmax/nm (ε/M−1 cm−1) 377 (1.57 × 104),
4610 | Org. Biomol. Chem., 2013, 11, 4602–4612
This journal is © The Royal Society of Chemistry 2013

View Article Online
Organic & Biomolecular Chemistry
Paper
355 (1.77 × 104), 323 (2.58 × 104), 278 (3.93 × 104), 252 (4.03 × (3.19 mL) in water (10.9 mL) at room temperature. Purified,
104); HRMS (ESI) m/z: ([M − 3Cl])3+ calcd for C46H38N8O3Ir3+, reduced iso-1 cytochrome c (150 nmol) and tris(2-carboxyethyl)-
314.4232; found, 314.4225; MS (ESI) m/z: ([M − 3Cl])3+, 314.15.
phosphine (75 nmol) was then added, and the reaction
Prior to bioconjugation, Ir(III) 4 was exchanged with chloride mixture left to stir in the dark for 19 h before concentration,
salt to increase solubility and yield.
dialysed into water and purified via immobilised metal affinity
chromatography (IMAC, Ni2+) using a gradient from 0 to
125 mM imidazole in sodium dihydrogen phosphate (20 mM),
[Ru(tpy)2](PF6)2 (Ru(II) 5)17
A solution of 2,2′:6′,2′′-terpyridine (113.8 mg, 0.488 mmol) and sodium chloride (0.5 M), pH 7.0 in 3.2 mL at 0.5 mL min−1
.
ruthenium(III) trichloride hydrate (49.6 mg, 0.239 mmol) in Fractions containing product were pooled, concentrated, and
ethylene glycol (33 mL) was heated at 110 °C for 21 h. The dialysed into water to yield bioconjugate Ru(II)–cyt
c
solution was then diluted with water (150 mL), filtered (40.1 nmol, 27%). MS (MALDI) m/z: 13 556 ([M − 2Cl])+,
through celite and the product was precipitated with requires 13 559.
ammonium hexafluorophosphate. The solid was collected by
centrifugation, washed with water and recrystallised with
Ir(III)–cyt c bioconjugate
acetonitrile/diethyl ether and collected by filtration and A solution of Ir(III) 4 (1.04 mg, 1000 nmol) in water (7.86 mL)
washed with acetonitrile yielding [Ru(tpy)2](PF6)2Ru(II) 5 as a was added to a solution of 94 mM sodium dihydrogen phos-
1
red solid (103.1 mg, 50%). H NMR (300 MHz, CD3CN) δ 8.73 phate, 94 mM ethylenediaminetetraacetic acid, pH 7.0
(d, J = 8.1 Hz, 4H), 8.48 (dt, J = 8.1, 1.0 Hz, 4H), 8.40 (t, J = (2.13 mL) at room temperature. Purified, reduced iso-1 cyto-
8.3 Hz, 2H), 7.91 (td, J = 7.8, 1.5 Hz, 4H), 7.32 (dq, J = 5.6, chrome
c (100 nmol) and tris(2-carboxyethyl)phosphine
0.8 Hz, 4H), 7.19–7.11 (m, 4H). MS (ESI) m/z: ([M − 2PF6]2+) (50 nmol) was then added, and the reaction mixture left to stir
calcd for C30H22N6Ru2+, 284.05; found, 283.80. These results in the dark for 23 h before being concentrated, dialysed into
are in agreement with those reported in the literature.17
water, and purified using the IMAC procedure applied to
Ru(II)–cyt c. Two separate fractions containing product were
concentrated and dialysed into water to yield bioconjugate
[Ir(tpy)(4′-(4-hydroxymethylphenyl-tpy)](PF6)3 (Ir(III) 6)
[Ir(tpy)]Cl3 1 (100 mg, 0.183 mmol) salt and 4′-(4-hydroxy- Ir(III)–cyt
c (33.0 nmol, 33%). MS (MALDI) m/z: 13 658
methylphenyl)-2,2′:6′2′′-terpyridine43 (67 mg, 0.198 mmol) ([M − 2Cl)]+, requires 13 650; 13 422 ([M − 2Cl − tpy])+ requires
were crushed together with a glass rod. Ethylene glycol 13 417; 13 230 ([M − 2Cl − tpy − Ir])+ requires 13 225.
(15 mL) was added and the mixture degassed with three
Enzyme induced polymersomes
freeze–thaw cycles before being heated to 160 °C in the dark
and under nitrogen for 20 min. The reaction mixture was In a typical experiment, a 1 mg mL−1 solution of PS140-b-PAA48
allowed to cool to room temperature before an aqueous solu- in tetrahydrofuran (33 μL) was injected into an enzyme/bio-
tion of excess ammonium hexafluorophosphate was added. conjugate solution (7.5 μM, 200 μL) in phosphate buffered
The resulting precipitate was collected by filtration and saline (150 mM, pH 7.2). The solution was allowed to equili-
washed with water, ethanol and diethyl ether, followed by brate for at least 24 h and extensively dialysed against water
recrystallisation from acetonitrile/diethyl ether. This was then using a 50 kDa molecular weight cut-off membrane over 24 h
purified by column chromatography (silica, 70 : 29 : 1, CH3CN : to remove non-encapsulated enzymes.
H2O : KNO3 (saturated)), followed by precipitation using
aqueous ammonium hexafluorophosphate, filtered, and recrys-
Photo-induced electron transfer studies
tallised from acetonitrile/diethyl ether to yield Ir(III) 6 as a Room temperature photo-induced electron transfer measure-
1
yellow solid (195 mg, 88%). Mp >300 °C; H NMR (300 MHz, ments of a non-covalent mixture of Ru(II) 5 and cytochrome c
CD3CN): δ 9.07 (s, 2H), 8.85 (d, 2H, J = 8.8 Hz), 8.80–8.70 (m, and bioconjugate were conducted in specialised small volume
3H), 8.59 (dd, 4H, J = 7.5, 0.7 Hz), 8.26–8.18 (m, 6H), 7.77 (d, quartz cuvettes designed for protein samples, allowing com-
2H, J = 7.7 Hz), 7.69 (dd, 2H, J = 5.7, 0.8 Hz), 7.58 (dd, 2H, J = plete exposure to irradiation with a constant area for all experi-
4.3, 0.5 Hz), 7.52–7.45 (m, 4H), 4.82 (s, 2H); 13C (75 MHz, ments (1.0 cm × 0.3 cm). A solution (80 μL or 120 μL) of 5 mM
CD3CN): δ 159.0, 157.0, 155.7, 155.5, 154.4, 154.3, 144.7, 143.9, sodium dihydrogen phosphate buffer, 5 mM ethylenediamine-
134.8, 130.8, 129.4, 128.7, 128.5, 127.6, 125.1, 64.0; IR (ATR): tetraacetic acid, pH 7.0 was prepared containing either a
νmax/cm−1 3418 (br), 3063 (w), 2939 (w), 2865 (w), 1606 (s), 1 : 1 mixture of cytochrome c (2.3 μM) and Ru(II) 5 (2.3
1548 (m), 1474 (m), 1403 (m), 1241 (m), 1105 (m), 1052 (m), 0.1 μM) or bioconjugate (2.3 μM) in bulk or membrane encap-
836 (s) (PF6); HRMS (ESI) m/z: ([M − PF6])+ calcd for sulated samples. Prior to irradiation, cuvettes were degassed
+
C37H28F12IrN6OP2 , 1055.1237; found, 1055.1226; MS (ESI) for 30 min at 0 °C under reduced pressure (120 mbar) and
m/z: ([M − PF6])+, 1055.21.
Ru(II)–cyt c bioconjugate
overlayed with nitrogen in the dark. Samples were irradiated in
a nitrogen purged UV-Vis spectrometer with a 465 nm or
372 nm light (LED) source for Ru(II) or Ir(III) bioconjugates,
A solution of Ru(II) 4 (0.9 mg, 975 nmol) in acetonitrile respectively. The LED was placed 2.5 cm from sample and cyto-
(600 μL) was added to a solution of 94 mM sodium dihydrogen chrome c reduction was monitored by UV absorbance at
phosphate, 94 mM ethylenediaminetetraacetic acid, pH 7.0 550 nm.
This journal is © The Royal Society of Chemistry 2013
Org. Biomol. Chem., 2013, 11, 4602–4612 | 4611

View Article Online
Paper
Organic & Biomolecular Chemistry
Stern–Volmer studies
16 I. Hamachi, T. Matsugi, S. Tanaka and S. Shinkai, Bull.
Chem. Soc. Jpn., 1996, 69, 1657.
Fluorescence emission measurements were made in special-
ised small volume quartz cuvettes designed for protein
samples. Fluorescence measurements were conducted in a
mixture of Ir(III) 6 (5 μM) with iso-1 cytochrome c (0 to 50 μM)
in an aqueous potassium nitrate solution (0.1 M) to maintain
constant ionic strength in a total volume of 130 μL. Measure-
ments were performed at 25 and 35 °C.
17 H. Hofmeier, P. R. Andres, R. Hoogenboom, E. Herdtweck
and U. S. Schubert, Aust. J. Chem., 2004, 57, 419.
18 J.-P. Collin, I. M. Dixon, J.-P. Sauvage, J. A. G. Williams,
F. Barigelletti and L. Flamigni, J. Am. Chem. Soc., 1999, 121,
5009.
19 M. Maestri, N. Armaroli, V. Balzani, E. C. Constable and
A. M. W. C. Thompson, Inorg. Chem., 1995, 34, 2759.
20 H. Hofmeier and U. S. Schubert, Chem. Soc. Rev., 2004, 33,
373.
Acknowledgements
21 J. Hovinen, Bioconjugate Chem., 2007, 18, 597.
22 K. Lashgari, M. Kritikos, R. Norrestam and T. Norrby, Acta
Crystallogr., Sect. C: Cryst. Struct. Commun., 1999, 55, 64.
23 E. C. Constable, J. Lewis, M. C. Liptrot and P. R. Raithby,
Inorg. Chim. Acta, 1990, 178, 47.
24 D. Keilin, Proc. R. Soc. London, Ser. B, 1930, 106, 418.
25 D. E. Shafer, J. K. Inman and A. Lees, Anal. Biochem., 2000,
282, 161.
We thank the Mark Wainwright Analytical Centre (The Univer-
sity of New South Wales) for providing access to instruments.
We thank the Australian Synchotron Facility for access to the
beamline. This work was supported by the Australian Research
Council
Discovery
Project
Grants
(DP0666325
and
DP120101604) and a Future Fellowship to PT (FT120100101).
We would also like to thank the UNSW for a scholarship to DH.
26 I. Sánchez-Barragán, J. M. Costa-Fernández, A. Sanz-Medel,
M. Valledor and J. C. Campo, Trends Anal. Chem., 2006, 25,
958.
27 J. R. Lakowicz, Principles of Fluorescence Spectroscopy,
Kluwer Academic/Plenum Press, New York, 2nd edn, 1999.
28 P. Thordarson, Chem. Soc. Rev., 2011, 40, 1305.
29 W. Leslie, A. S. Batsanov, J. A. K. Howard and J. A. Gareth
Williams, Dalton Trans., 2004, 623.
Notes and references
1 G. T. Hermanson, Bioconjugate Techniques, Elsevier Inc.,
San Diego, CA, 2nd edn, 2008.
2 J. Wang, Chem. Rev., 2008, 108, 814.
30 D. Hvasanov, J. Wiedenmann, F. Braet and P. Thordarson,
Chem. Commun., 2011, 47, 6314.
31 R. A. Marcus and N. Sutin, Biochim. Biophys. Acta, 1985,
811, 265.
32 H. B. Gray and J. R. Winkler, Annu. Rev. Biochem., 1996, 65,
537.
33 N. S. Hush, Trans. Faraday Soc., 1961, 57, 557.
34 M. Cordes and B. Giese, Chem. Soc. Rev., 2009, 38, 892.
35 J. R. Winkler, T. L. Netzel, C. Creutz and N. Sutin, J. Am.
Chem. Soc., 1987, 109, 2381.
36 G. Steinberg-Yfrach, P. A. Liddell, S.-C. Hung, A. L. Moore,
D. Gust and T. A. Moore, Nature, 1997, 385, 239.
37 G. Steinberg-Yfrach, J.-L. Rigaud, E. N. Durantini,
A. L. Moore, D. Gust and T. A. Moore, Nature, 1998, 392, 479.
38 H.-J. Choi and C. D. Montemagno, Nano Lett., 2005, 5,
2538.
39 J. R. Peterson and P. Thordarson, Chiang Mai J. Sci., 2009,
26, 236.
40 Bruker, APEX2 Suite, Bruker AXS Inc., Madison, WI, USA,
2007.
3 I. Willner, Science, 2002, 298, 2407.
4 I. Willner, Y.-M. Yan, B. Willner and R. Tel-Vered, Fuel
Cells, 2009, 9, 7.
5 K. Ulbrich, J. Strohalm, V. Subr, D. Plocova, R. Duncan and
B. Rihova, Macromol. Symp., 1996, 103, 177.
6 J. R. Peterson, T. A. Smith and P. Thordarson, Chem.
Commun., 2007, 1899.
7 J. R. Peterson, T. A. Smith and P. Thordarson, Org. Biomol.
Chem., 2010, 8, 151.
8 J. P. Sauvage, J. P. Collin, J. C. Chambron, S. Guillerez,
C. Coudret, V. Balzani, F. Barigelletti, L. De Cola and
L. Flamigni, Chem. Rev., 1994, 94, 993.
9 S. Mayo, W. Ellis, R. Crutchley and H. Gray, Science, 1986,
233, 948.
10 F. Millett and B. Durham, Methods Enzymol., 2009, 456, 95.
11 M. Heller and U. S. Schubert, Eur. J. Org. Chem., 2003, 947.
12 D. C. Goldstein, Y. Y. Cheng, T. W. Schmidt, M. Bhadbhade
and P. Thordarson, Dalton Trans., 2011, 40, 2053.
13 X.-J. Yang, F. Drepper, B. Wu, W.-H. Sun, W. Haehnel and
C. Janiak, Dalton Trans., 2005, 256.
41 G. Sheldrick, Acta Crystallogr., Sect. A: Foundam. Crystal-
logr., 2008, 64, 112.
14 S. Hahm, B. Durham and F. Millett, Biochemistry, 1992, 31,
3472.
42 W. Kabsch, J. Appl. Crystallogr., 1993, 26, 795.
43 A. Winter, C. Ulbricht, E. Holder, N. Risch and
U. S. Schubert, Aust. J. Chem., 2006, 59, 773.
15 K. Wang, H. Mei, L. Geren, M. A. Miller, A. Saunders,
X. Wang, J. L. Waldner, G. J. Pielak, B. Durham and
F. Millett, Biochemistry, 1996, 35, 15107.
4612 | Org. Biomol. Chem., 2013, 11, 4602–4612
This journal is © The Royal Society of Chemistry 2013