An efficient route to asymmetrically diconjugated tris(heteroleptic) complexes of Ru(ii)

Article abstract of DOI:10.1039/c6ra06086b

A highly efficient and versatile route to the preparation of tris(heteroleptic) Ru(ii) polypyridyl complexes is described which permits access to two or more independently conjugatable termini in the final structure. The strategy utilizes the well-known R

Full text of DOI:10.1039/c6ra06086b

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An Efficient Route to Asymmetrically Diconjugated
tris(heteroleptic) Complexes of Ru(II)
Christopher S. Burke a and Tia E. Keyesa
Received 00th January 20xx,
Accepted 00th January 20xx
A highly efficient and versatile route to preparation of tris(heteroleptic) Ru(II) polypyridyl complexes is described which
permits access to two or more independently conjugatable termini in the final structure. The strategy utilizes the well-
known Ru(DMSO)4Cl2 precursor to form the Ru(N^N)(DMSO)2Cl2 product and then proceeds through an oxalate
intermediate which can be cleaved under acidic conditions to control the stoichiometric addition of polypyridyl ligands to
the Ru(II) coordination sphere enabling the stepwise assembly of each heteroleptic complex. To exemplify this approach,
three complexes were prepared including the novel: [Ru(dppz)(bpyArCOOH)(bpyArCOOEt)]2+ (where dppz is
dipyridophenazine, bpyArCOOH and bpyArCOOEt are 4-(4-carboxyphenyl)- and 4-(4-ethoxycarbonylphenyl)- 2,2-bipyridine
respectively) in which the synthetic yield from the RuCl3 starting material to final product is 82 %. A sequential
conjugation-deprotection-conjugation step is then described to yield a Ru(II) complex which is both PEGylated and
peptide-conjugated. This synthetic approach offers a useful tool to expand the structural diversity of bis coordinated Ru(II)
polypyridyl complexes and provides a simple route to building multifunctionalility into such complexes which should
broaden their application in particular in the domain of bioimaging and therapy.
DOI: 10.1039/x0xx00000x
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well-established preparation of the [Ru(N^N)2Cl2] intermediate
with subsequent conversion to tris-chelates.10
Introduction
Indeed,
synthesis through the dichloride intermediate can itself be
problematic due to formation of the intractable side product
[Ru(N^N)2(CO)Cl]+ which until a recent approach reported by
Rau et al., blocked chelation of the heteroligand.11
The wide utility of luminescent Ru(II) polypyridyl complexes
has been demonstrated for decades across diverse photonics
applications ranging from dye sensitised solar cells and
photocatalysis1–4 to the more recent burgeoning fields of bio-
imaging and metal-based theranostics.5–7 As the breadth of
application of this family of complexes grows so does the
demand for greater structural versatility. Although synthetic
routes to asymmetric ligand coordination is well established
for tridentate ligands related to the 2,2’:6’,2’’-terpyridine
The prevalence of the [Ru(N^N)2Cl2] synthetic route to tris
bis-chelated ruthenium complexes has limited somewhat the
structural diversity of the synthetic library of Ru(II) polypyridyl
complexes. And, as the application areas of such complexes
expands, particularly in biological domains there is a growing
demand to create structures which have multiple
independently modifiable functionalities as shown in Figure 1.
This can be achieved through preparation of asymmetric tris-
heteroleptic metal-ligand systems. Current approaches to
forming tris-heteroleptic bidentate Ru(II) chelates of the form,
ligand.8,9
A drawback of such complexes is that their
luminescence intensities and lifetime are often relatively low,
and this limits their application, particularly in bioimaging
applications. The bis-chelate polypyridyl complexes of Ru(II)
offer often excellent photophysical properties but the
structural diversity of these complexes is largely limited to
constructs of the general form: [Ru(N^N)2(N^N)’]2+ (where
N^N is an N-donor bidentate polypyridyl ligand). Generally
within this model, often it is the ternary ligand (N^N)’ that is
varied to impart specific functionality, with the ‘bis-ligand’
(N^N) most often one of either 2,2’-bipyridine (bpy) or 1,10-
phenanthroline (phen). Consequently, the route to the
synthesis of such complexes is almost universally through the
[Ru(N^N)(N^N)’(N^N)’’]2+
(Figure
1);
include;
(i)
decarbonylation using photolysis or otherwise from for
example [Ru(CO)2Cl2]n precursor, (ii) cyclometalation from Ru-
cymene type starting materials (iii) the use of a Ru(DMSO)4Cl2
intermediate.12–17 Beyond this, one-pot syntheses and solid
Function 2
N
Function 1
N
N
N
Ru2+
N
N
Function 3
Figure 1 – Schematic illustrating the diversity in functionality that
may be incorporated into Ru(II) tris heteroleptic chelate complexes.
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phase methods have also been reported.18,19 However, the
complexity, synthetic yields and/or generality of these routes
are frequently limiting. For example, to the best of our
knowledge, the highest yield reported to date was the 5-step
procedure reported by Myahkostupov and Castellano who
implemented the original protocol described by Mann et al. to
provide a Ru(II) tris heteroleptic complex from [Ru(Bz)Cl2]2
precursor in 61 % yield.13,17 Herein, we expand the synthetic
diversity of bis-chelated Ru(II) complexes through a complete,
quite general and high yielding route to Ru(II) tris-heteroleptic
complexes.
Results and Discussion
Synthesis
The route exploited here to produce Ru(II) polypyridyl tris-
heteroleptic complexes in high yield is shown in Scheme 1. We
adopted Ru(DMSO)4Cl2 as a Ru(II) precursor for the stepwise
addition of successive bidentate polypyridyl ligands to the
coordination sphere. In our hands, the original synthesis
reported by Evans et al.20 was found to be somewhat
inconsistent in terms of the yield and isomeric purity of the
products. Consequently, the approach reported by Alston et
al.38 was used as this route produced isomerically pure cis,fac-
RuCl2(κS-DMSO)3(κO-DMSO) in quantitative yield (98 %). The
ability to efficiently displace two DMSO ligands from this
precursor and coordinate a single bidentate polypyridyl ligand
has been widely reported.20–22 The ligand of choice in the
present case was dipyrido[3,2-a:2',3'-c]phenazine (dppz) – as
this ligand is of significant broad interest in ruthenium
chemistry due to its capacity to bind DNA and the “solvent
switch” effect it induces in complexes of the type;
[Ru(bpy/phen)2(dppz)]2+.39,40 Such complexes exhibit virtually
no emission in aqueous media but emit strongly upon
protection of the phenazine moiety from hydrogen bonding
for example on its intercalation in DNA or a lipid membrane.28
The Ru(II)-dppz solvate complex, Ru(dppz)(DMSO)2Cl2 (1); was
obtained quantitatively (99 %) under simple ethanolic reflux
and structurally characterised by NMR, mass spectrometry and
The Ru(DMSO)4Cl2 intermediate is perhaps the most
straight-forward route to bis-chelates and was adapted for
optimisation as part of the present work. It has been widely
demonstrated that stoichiometric addition of a ligand to
Ru(DMSO)4Cl2 yields Ru(N^N)(DMSO)2Cl2 in high yield.20–22
Further substitution may then proceed via the classic
Ru(N^N)2Cl2 route with subsequent ternary chelation in
aqueous alcohol. Although well established, this approach
frequently leads to poor synthetic yields and, as the dichloride
intermediate can be difficult to obtain in a pure form due to
by-products such as tris-chelates and, as described
[Ru(N^N)2(CO)Cl]+, this method frequently requires significant
follow on purification.23 We rationalized that both yield and
purity may be improved by avoiding the formation of the
dichloride intermediate. Instead we propose the use of an
oxalate as an intermediate since their chemistry with Ru(II) has
been well studied and they are synthetically easily accessible.
Furthermore, oxalate ligand can be hydrolysed in acidic
conditions to permit ternary ligand chelation.24–26 Combining
the use of Ru-DMSO and Ru-oxalate intermediates, we
describe three examples of the successful implementation of
microanalyses (see ESI†). Analysis of the number of aromatic
signals in the 1H NMR spectrum indicates asymmetry in the
complex indicating that both of the DMSO ligands and the
chlorides conform to a cis-configuration relative to one
another.
this
route
to
convert
RuCl3.nH2O
to
pure
[Ru(N^N)(N^N)’(N^N)’’]2+ with unprecedented yield, in excess
of 82 % in each case start-to-finish.
In accordance with the most widely accepted route to
preparation of tris Ru(II) polypyridyl chelates, i.e. via a
Ru(N^N)2Cl2 intermediate, we first attempted the addition of
bpy to Ru(dppz)(DMSO)2Cl2 (1) to yield Ru(dppz)(bpy)Cl2. This
route, via reflux in LiCl/DMF10 or in ethylene glycol23 was found
to be inefficient and the crude isolates in each case typically
contained significant quantities of unreacted precursor or
We have devised this approach in the context of our
interest in developing receptor-directed Ru(II) polypyridyl
complexes for imaging of sub-cellular structures in live cells.27–
31
Indeed, the application of transition metal luminophores to
cell imaging is a rapidly growing field of investigation.32–34 The
use of conjugated biomolecules such as peptides or polymeric
moieties such as PEG have been demonstrated to improve
solubility, reduce cytotoxicity and impart membrane
permeability and targeting to metal complex cargo.35–37 The
incorporation of a conjugatable terminus within the metal
complex is a prerequisite to progress in this area. In the Ru(II)
conjugates reported to date, complexes of the form;
[Ru(N^N)2(N^N)’]2+ have been applied where the complex is
conjugated at an appropriate functional group at the ternary
ligand (N^N)’ or far less commonly identical groups bound to
both of the ‘bis-ligands’ (N^N). However, using the approach
presented here the incorporation of one, two or three
conjugations as indicated in Figure 1 is possible and indeed the
implementation of different conjugation termini is easily
achieved. In our exemplar here, we describe a synthetic route
to a novel dual conjugate which is both PEGylated and peptide
conjugated.
under
longer
reaction
times,
the
tris-chelate,
[Ru(dppz)(bpy)2]2+; as the majority product. Furthermore,
purification of the dichloride was not trivial and impacted on
the final yield significantly. A cleaner, more controlled
approach was therefore developed which employed dioxane
as solvent under reflux with a donor solvent added to catalyse
the ligand substitution. The lower reflux temperature and
poorer donor ability of dioxane made it easier to limit extent
of reaction by controlling water content. Adjustment of the
reaction mixture up to
Ru(dppz)(bpy)Cl2 in 78
5
%
v/v water provided pure
%
yield after simple filtration.
Unfortunately, the method was found to be quite specific to
this ligand system and the analogous Ru(dppz)(bpyArCOOH)Cl2
complex was never isolated by this route.
The lack of success by these standard and modified routes
to a generalised synthesis of an asymmetric bis-polypyridyl
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Scheme 1 – Synthesis of the Ru(II) tris heteroleptic complexes via Ru-DMSO and Ru-oxalate intermediates.
intermediate prompted us to seek an alternative to the structurally characterised by NMR, elemental analysis and
dichloride intermediate that could be obtained in high yield mass spectroscopy which confirmed their identity and purity.
and purity and crucially, which is reactive enough for As Scheme 1 indicates the tris-heteroleptic complexes were
subsequent ternary chelation. Ru(II)-oxalates have been well obtained with exceptional yields with overall in each case of
studied across the literature though they have rarely been more than 82 % from the ruthenium chloride precursor to the
used as synthetic precursors to tris-chelated Ru(II) final purified product.
complexes.24–26 This is despite their advantages of being less
labile than the chloride and incapable of deviation from the Conjugation – Exploiting asymmetry to expand functionality
desired cis-type configuration within the Ru(II) coordination
To demonstrate the practicality of this approach as a route to
sphere. Herein, Ru(dppz)(DMSO)2Cl2 (1) was converted to
complexes
with
mixed
conjugation,
[Ru(dppz)(bpyArCOOH)(bpyArCOOEt)]2+ was subjected to the
functionalizations shown in Scheme 2. The complex was
prepared so that it contains both a free and protected acid at
its periphery in order to enable successive coupling to different
vectors.
Ru(dppz)(bpy)(ox) (2) and Ru(dppz)(bpyArCOOH)(ox) (3) in >
94 % yield in each case following a facile two-stage reflux with
isolation of the product in each case by simple filtration. Both
oxalate complexes were characterised by 1H NMR, mass
spectrometry and microanalyses which confirm their
successful preparation. Critically, the oxalate ligand hydrolyses
rapidly at low pH to provide Ru(II) solvates.41 In our hands, the
bright orange mixed aquo/acetonitrile solvate was found to be
most easily accessible by treatment of a suspension of the Ru-
oxalate with aqueous 1 M HClO4 in acetonitrile. Crucially, the
solvate complex precipitates in water as the perchlorate salt,
permitting its isolation by filtration after which the solid was
washed to eliminate residual acid.
PEGylation is often performed to improve aqueous
solubility and reduce cytotoxicity as well as improve cell
uptake of a conjugate.35,42 Signal peptides can be coordinated
to direct cargo to specific organelles within the cell e.g. in the
case of imaging to permit study of dynamic cellular
processes.31,43 Therefore, in an imaging application it is useful
to be able to combine both PEGylation with a targeting agent
at a single probe. Herein, using the tris-heteroleptic complex
(6) we prepared a metal complex luminophore that is both
signal peptide conjugated and PEGylated. First, the free acid
was exploited for amide conjugation to an amine terminated
PEG15 chain using the well-known HBTU coupling chemistry.
The ester was then de-protected through a facile hydrolysis
using LiOH and was followed by acid work up to provide
another free acid terminus. Since discrete PEG chains are
employed, both the ester and acid Ru-PEG complexes could be
unequivocally characterised by 1H NMR and mass spectroscopy
Reaction of the bis-chelated Ru(II) solvates in ethylene
glycol with stoichiometric quantities of ligand yielded the
target tris-heteroleptic complexes in yields in excess of 86 %
from the oxalate precursor following conventional flash
column chromatography on silica. The resulting novel
constructs;
[Ru(dppz)(bpy)(bpyArCOOEt)](PF6)2
(5)
(4)
,
[Ru(dppz)(bpy)(bpyArCOOH)](PF6)2
and
[Ru(dppz)(bpyArCOOEt)(bpyArCOOH)](PF6)2 (6) were obtained
as a mixture of isomers as indicated by their respective 1H
- +
NMR and COSY spectra (see ESI†). The compounds were fully
where the molecular ion corresponding to the [M – PF6 ] ion
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and the ruthenium isotope pattern are clearly evident (see peptide conjugation was also confirmed by UV-vis
ESI ). spectroscopy and by HPLC where the elution time of the
†
Scheme 2 – Synthesis of the PEGylated and peptide-conjugated Ru(II)-dppz construct.
The nuclear localising signal (NLS) VQRKRQKLMP–NH2 was diconjugate on reverse phase HPLC was extended to 9.93 min
then conjugated to the newly de-protected acid site. This compared to the parent (4.84 min) or monoconjugates (8.36
peptide is derived from the transcription factor NF-kB, which min and 8.65 min for the ester and acid respectively). The
functions to internalise NF-kB into the nucleus, it has purity of all of the conjugates was also confirmed by HPLC to
previously been reported as a transmembrane molecular cargo be > 98 % purity in each case.
carrier.29 The NLS was conjugated to the Ru-PEG conjugate
again through a HBTU coupling protocol. The resulting Photophysical characterisation
diconjugate complex: [Ru(dppz)(bpy-Ar-PEG)(bpy-Ar-NFkB)]6+
Preliminary spectroscopic and photophysical characterisation
was purified on silica plates and obtained as the
of the complexes and conjugates was carried out for
hexafluorophosphate salt. The 1H NMR spectrum of the
completeness. We were interested in particular to explore the
diconjugate exhibits signals that correspond to the Ru(II) core,
impact, if any, that the conjugated peptide and PEG had on the
the PEG chain and also multiple signals in the aliphatic region
photophysical properties of the Ru-dppz core in aqueous
attributed to the short peptide sequence. The relative
media.
1
integration in HNMR was as expected for the ruthenium, PEG
The unconjugated probes all exhibit spectroscopic and
and peptide moieties for all but the exchangeable peptide
photophysical behaviour typical of Ru(II)-dppz complexes with
protons. Methanol-d4 was employed as solvent which
distinct ligand absorptions at ca. 280 and 350 nm and a broad
simplified the spectrum through deuterium substitution
MLCT band centred around 450 nm region (Table 1 and Figure
equilibria established with exchangeable protons on the
2). As expected, luminescence is observed in acetonitrile with
polypeptide backbone at arginine and lysine residues. The
emission maxima ca. 620 nm. The luminescence lifetime for
mass spectrum of the diconjugate (9) was markedly different
the parent complexes lies in the range 230 – 250 ns increasing
to that of the monoconjugate (8). Despite the absence of a
to ca. 400 ns upon deaeration under N2 purge. In all cases
molecular ion, evidence for the successful amidation coupling
there was no luminescence observed from the dppz containing
was deduced from two peaks at m/z values of 1753.58 and
complexes when dissolved in water and titration of the
1607.62 which are shifted slightly relative to (8) and are
luminescent
acetonitrile
solution
of
[Ru(dppz)(bpy)(bpyArCOOH)]2+ with water indicates that half
of the emission is quenched when the water content exceeds 5
% v/v and emission approaches the baseline above 15 %.
assigned to the conversion of the free acid of (8) to the amide
of (9) thus indicating fragmentation at the newly coupled
amide. Additionally, an overlay of the mass spectra of (8) and
(9) reveals new Ru isotope clusters in the m/z region of 800 to
1500, which are attributed to ions formed from extensive
fragmentation of the polypeptide backbone. Confirmation of
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0 – 15 %
H2O
(a)
(b)
As shown in Table 1, the luminescent lifetimes for the 400 and 100 MHz or 600 and 150 MHz respectively using
mono ester compound (4) decreases by about 10 ns on de- Bruker spectrometers and deuterated solvent for homo-
protection and the lifetime of (6) is higher than (4) or (5) but nuclear lock. The spectra were processed using Bruker Topspin
decreases modestly on conjugation to the PEG moiety, with no software and were calibrated against solvent peaks according
significant change on conjugation of the peptide. Luminescent to published values.46 Elemental analyses were performed at
quantum yields in aerated acetonitrile are similar for the Microanalytical Laboratory at University College Dublin.
complexes (4) – (6) and sit in the range 2.6 – 3.0 % which is High Resolution Mass Spectrometry (HR-MS) was performed at
typical of Ru(II)-dppz complexes of this type.5 As expected for the Mass Spectrometry Facility, University College Dublin or at
bis-chelated Ru(II) polypyridyl complexes, the complexes are the Mass Spectrometry Unit, Trinity College Dublin. The
stable with no apparent degradation of the complexes (4) – (6) syntheses of the Ru(II) complexes described below were
over the course of two months evident from UV-Vis performed under nitrogen and in the absence of light.
spectroscopy. In terms of the application of such conjugates to Preparative LC was performed using flash columns or plates as
imaging, the photophysical properties of the complex, indicated. Analytical HPLC was performed on a Varian 940-LC
including the light-switch effect is maintained on bis- Liquid Chromatograph using a Hichrom C18 column (4.6 x 250
conjugation.
mm). Gradient elution was employed in the separation using a
0.1% TFA in MeCN/Water mixture starting at 95/5 and
changing linearly to 50/50 over 20 minutes at 1.8 mL/min
flowrate. PDAD was used for peak detection and the analysis
was followed by monitoring 220 nm and 450 nm channels.
Experimental
General Information
cis-Ru(DMSO)4Cl2,38 dppz,44 bpyArCOOEt and bpyArCOOH45
were synthesised according to reported procedures. Discrete
methoxy-PEG15-amine, m-dPEG, was purchased from Quanta
Biodesign. Peptides (> 98 %) were procured from Celtek
Peptides, TN, USA. All other materials were obtained from
Sigma Aldrich Chemical Co. and were used without further
Synthesis of the Ru(II) parent complexes
[Ru(dppz)(DMSO)2Cl2] (1): cis-Ru(DMSO)4Cl2 (500 mg, 1.03
mmol) and dppz (290 mg, 1.03 mmol) were heated at reflux in
ethanol (35 mL) for 2 h. The reaction was then cooled to room
temperature and the solvent volume reduced to ca. 10 mL in
vacuo. The precipitate that forms upon cooling was filtered,
1
purification. H and 13C NMR spectra were recorded at either
Compound
Solvent
λ Absorbance (ε)
λ Emission
nm
τ
ns
φ lum
0.027
0.026
0.030
nm (x10-3 M-1 cm-1)
[Ru(dppz)(bpy)(bpyArCOOEt)]2+ (4)
[Ru(dppz)(bpy)(bpyArCOOH)]2+ (5)
[Ru(dppz)(bpyArCOOH)(bpyArCOOEt)]2+ (6)
[Ru(dppz)(bpyAr-PEG)(bpyArCOOEt)]2+ (7)
[Ru(dppz)(bpyAr-PEG)(bpyAr-NFkB)]6+ (9)
MeCN
H2O
282 (112.6), 355 (29.0), 454 28.1).
281 (86.1), 359 (20.3), 452 (18.5).
282 (94.8), 354 (24.0), 454 (23.2).
281 (68.8), 358 (18.3), 455 (17.1).
277 (72.9), 355 (19.4), 459 (19.6).
279 (73.3), 357 (20.2), 457 (20.0).
279 (78.7), 354 (20.1), 459 (19.9).
279 (73.0), 357 (20.7), 458 (19.1).
280 (76.8), 354 (20.1), 459 (19.6).
280 (66.5), 356 (18.8), 457 (17.1).
617
None
620
None
616
None
619
None
621
None
239 ± 1
MeCN
H2O
228 ± 1
253 ± 2
246 ± 2
249 ± 1
MeCN
H2O
MeCN
H2O
MeCN
H2O
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washed with minimal cold ethanol and copious amounts of
hexane/diethyl ether and dried under nitrogen. In general, the
product is isolated pure in this manner, otherwise extraction
into acetone and re-precipitation using ether/hexane yields
the pure Ru(II) DMSO-solvate. Yield: light-brown solid, 625 mg
the bpyArCOOR ligand (0.16 mmol) and heated at reflux for 4 –
6 h. The deep red mixture was cooled to room temperature
and
poured
on
stirring
aqueous
ammonium
hexafluorophosphate to precipitate the crude complex as the
hexafluorophosphate salt. The solids were filtered, washed
with water and dried under a nitrogen stream to afford the
1
(1.02 mmol, 99 %). H NMR (400 MHz, CDCl3) δ (ppm): 10.22
target complexes as
a mixture of geometric isomers.
(d, 1 H); 10.05 (d, 1 H); 9.79 (d, 1 H); 9.69 (d, 1 H); 8.43 (m, 2
H); 8.11 (t, 1 H); 8.05 (m, 2 H); 7.93 (t, 1 H); 3.65 (s, 3 H); 3.60
(s, 3 H); 3.26 (s, 3 H); 2.70 (s, 3 H). 13C NMR (100 MHz, CDCl3) δ
(ppm): 157.66, 154.26, 152.02, 150.162, 142.90, 139.58,
139.49, 134.89, 133.91, 132.04, 130.36, 129.88, 129.68,
126.10, 126.03, 47.20, 46.47, 45.49, 44.39. Anal. Calculated
(Found) for C22H22Cl2N4O2S2Ru: C 43.28 (43.78); H 3.63 (3.35);
Purification was performed on short silica flash columns using
90/10/1 MeCN/H2O/20% w/v KNO3 (aq). The product fraction
was concentrated in vacuo, precipitated using ammonium
hexfluorophosphate and filtered. The solids were taken up in
minimum acetone, filtered, concentrated and re-precipitated
by slow addition to stirring diethyl ether. Filtration yielded the
bright orange pure complexes as a mixture of geometric
isomers.
N
9.18 (9.26); Cl 11.61 (11.42). HR-MS (ESI-TOF) m/z:
Calculated for C22H22Cl2N4O2S2Ru [M]+: 609.9605; Found:
[Ru(dppz)(bpy)(bpyArCOOEt)](PF6)2 (4): Yield: orange
solid, 166 mg (0.146 mmol, 92 %). 1H NMR (600 MHz, CD3CN) δ
(ppm): 9.68 (m, 2 H); 8.80 (2x dd, 1 H); 8.73 (2x dd, 1 H); 8.56
(2x t, 2 H); 8.49 (m, 2 H); 8.12 – 8.25 (m, 8 H); 8.00 – 8.07 (m, 2
H); 7.91 (m, 5 H); 7.49 – 7.79 (2x m, 3 H); 7.29 – 7.48 (2x m, 3
H); 4.37 (2x q, 2 H); 1.38 (2x t, 3 H). 13C NMR (150 MHz, CD3CN)
δ (ppm): 166.56, 158.78, 158.57, 158.20, 158.17, 157.97,
157.92, 154.71, 154.63, 153.33, 153.16, 153.06, 153.01,
152.96, 151.47, 151.42, 149.41, 143.77, 141.01, 140.88,
140.78, 139.03, 138.94, 138.86, 134.56, 133.54, 133.20,
133.12, 131.89, 131.16, 131.09, 128.76, 128.69, 128.61,
128.50, 128.44, 128.39, 126.10, 125.91, 125.59, 125.53,
125.34, 125.28, 123.21, 123.15, 62.20, 14.48. Anal. Calculated
(Found) for C47H34N8O2P2F12Ru: C 49.79 (49.18); H 3.02 (2.68);
609.9604.
General Procedure for synthesis of [Ru(dppz)(N^N)(ox)]
Ru(dppz)(DMSO)2Cl2 (312 mg, 0.511 mmol) and sodium oxalate
(100 mg, 0.746 mmol) were heated at reflux in water (15 mL)
for 1 h. The reaction was then cooled to room temperature
and added to a hot solution of the polypyridyl ligand (0.511
mmol) in 15 mL ethylene glycol. The resulting mixture was
heated at reflux for 3 h, cooled to room temperature and then
added dropwise to 50 mL of stirring water. After 30 minutes,
the precipitates were filtered through a 0.4 μm membrane.
The solids were washed with copious amounts of water and
minimal acetone before drying thoroughly under a nitrogen
stream.
N
9.88 (9.59). HR-MS (ESI-TOF) m/z: Calculated for
C45H30N8O2PF6Ru [M - PF6]+: 989.1490; Found: 989.1477.
[Ru(dppz)(bpy)(bpyArCOOH)](PF6)2 (5): Yield: orange solid,
148 mg (0.134 mmol, 86 %). 1H NMR (600 MHz, CD3CN) δ
(ppm): 9.68 (m, 2 H); 8.81 (2x dd, 1 H); 8.73 (2x d, 1 H); 8.56
(2x t, 2 H); 8.48 (m, 2 H); 8.22 (m, 3 H); 8.14 (m, 5 H); 8.04 (m,
2 H); 7.84 – 7.97 (m, 5 H); 7.54 – 7.76 (m, 3 H); 7.28 – 7.49 (m,
3 H). 13C NMR (150 MHz, CD3CN) δ (ppm): 168.65, 158.68,
158.47, 158.26, 158.21, 158.04, 157.99, 157.96, 154.71,
154.66, 153.21, 153.15, 153.07, 152.98, 151.49, 143.77,
141.02, 139.00, 138.93, 138.84, 134.53, 133.53, 131.88,
131.35, 131.28, 130.62, 128.71, 128.61, 128.57, 128.49,
128.44, 128.25, 128.17, 126.03, 125.85, 125.60, 125.54,
125.34, 125.27, 123.13, 123.08. Anal. Calculated (Found) for
C45H30N8O2P2F12Ru: C 48.88 (49.41); H 2.73 (2.58); N 10.13
(10.10). HR-MS (ESI-TOF) m/z: Calculated for C45H30N8O2PF6Ru
[M - PF6]+: 961.1183; Found: 961.1190.
[Ru(dppz)(bpy)(ox)] (2): Yield: purple-black fine powder,
1
313 mg (0.499 mmol, 98 %). H NMR (600 MHz, DMSO-d6) δ
(ppm): 9.62 (d, 1 H); 9.32 (d, 1 H); 9.26 (d, 1 H); 9.00 (d, 1 H);
8.83 (d, 1 H); 8.67 (d, 1 H); 8.53 (d, 1 H); 8.47 (d, 1 H); 8.36 (dd,
1 H); 8.22 (q, 1 H); 8.15 (m, 3 H); 7.92 (t, 1 H); 7.80 (t, 1 H); 7.70
(m,
2 H); 7.12 (t, 1 H). Anal. Calculated (Found) for
C30H18N6O4Ru.2H2O: C 54.70 (54.30); H 2.89 (3.34); N 12.72
(12.66). HR-MS (ESI-TOF) m/z: Calculated for C30H18N6O4RuNa
[M + Na]+: 651.0325; Found: 651.0358.
[Ru(dppz)(bpyArCOOH)(ox)] (3): Yield (from 200 mg Ru(II)
1
starting material): black solid, 231 mg (0.308 mmol, 94 %). H
NMR (400 MHz, DMSO-d6) δ (ppm): 13.16 (br s, 1 H, COOH);
9.61 (d, 1 H); 9.33 (d, 1 H); 9.26 (d, 1 H); 9.11 (d, 1 H); 9.01 (2s,
2 H); 8.51 (d, 1 H); 8.46 (d, 1 H); 8.37 (dd, 1 H); 8.26 (m, 1 H);
8.15 (m, 3 H); 8.03 (s, 4 H); 7.94 (t, 1 H); 7.76 (d, 1 H); 7.73 (dd,
1
H); 7.50 (dd, 1 H). Anal. Calculated (Found) for
C37H22N6O6Ru.H2O: C 58.04 (57.50); H 3.16 (2.98); N 10.98
(11.17).
[Ru(dppz)(bpyArCOOH)(bpyArCOOEt)](PF6)2 (6): The crude
product was obtained using an identical procedure to that
described for (4) and (5). Purification was performed on silica
General Procedure for [Ru(dppz)(bpy)(bpyArCOOR)](PF6)2
Ru(dppz)(bpy)(ox) (100 mg, 0.159 mmol) was suspended in 2 using 70/26/4/2 CHCl3/MeOH/H2O/AcOH. The concentrated
mL acetonitrile and 2 mL of 1 M perchloric acid was added. product
fraction was treated with aqueous
After refluxing for 1 h, a red-brown solution of the Ru-solvate hexafluorophosphate to precipitate the product salt which was
was obtained and after cooling it was poured on 10 mL stirring filtered. Dissolution in minimum acetone and re-precipitation
water. The solids that precipitated were filtered and dried from diethyl ether yielded the final complex as a mixture of
yielding the crude burnt-orange bis-solvated Ru(II) complex. isomers. Yield: orange solid, 152 mg (0.121 mmol, 91 %). 1H
The intermediate was dissolved in ethylene glycol (10 mL) with NMR (600 MHz, CD3CN) δ (ppm): 9.67 (m, 2 H); 8.80 (m, 4 H);
6 | J. Name., 2012, 00, 1-3
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8.46 (m, 2 H); 8.28 (t, 1 H); 8.24 (m, 1 H); 8.10 – 8.22 (m, 7 H); (s, 3 H, PEG –OCH3). HR-MS (MALDI-QTOF) m/z: Calculated for
8.07 (m, 1 H); 8.01 (d, 1 H); 7.98 (t, 1 H); 7.89 – 7.96 (m, 5 H); C83H97N9O18PF6Ru [M - PF6]+: 1754.5637; Found: 1754.5691.
7.86 (d, 1 H); 7.81 (m, 2 H); 7.75 (qd, 1 H); 7.55 (m, 1 H); 7.52
(m, 1 H); 7.32 (t, 1 H); 4.37 (2x q, 2 H); 1.38 (2x t, 3 H). 13C NMR [Ru(dppz)(bpy-Ar-PEG)(bpy-Ar-NFkB)](PF6)6
(9): A DMF (1
(150 MHz, CD3CN) δ (ppm): 166.55, 166.51, 158.26, 154.75, mL) solution of (8) (5 mg, 0.006 mmol) and HBTU (12 mg,
154.67, 153.30, 151.48, 149.90, 143.76, 140.99, 140.89, 0.031 mmol) was prepared and to this stirring solution at room
138,99, 138.90, 134.58, 133.52, 131.90, 131.33, 131.27, temperature was added DIPEA (10 uL, 0.06 mmol). After 15
131.14, 131.09, 130.62, 128.71, 128.62, 128.49, 128.24, minutes, a solution of the short peptide in DMF (5 mg in 0.2
128.17, 125.95, 125.89, 125.63, 123.23, 123.17, 123.07. Anal. mL, 0.007 mmol) was added to the reaction mixture. The
Calculated (Found) for C54H38N8O4P2F12Ru: C 51.72 (51.11); H solution was allowed to stir for 24 h and was then treated with
3.05 (3.05); N 8.94 (8.59). HR-MS (ESI-TOF) m/z: Calculated for diethyl ether to precipitate a crude mass. The suspension was
C54H38N8O4PF6Ru [M - PF6]+: 1109.1701; Found: 1109.1757.
poured on a short flash column (RP-C18) and the reaction
solvent rinsed free using acetonitrile (0.1 % TFA) as eluent.
Residual starting material is first eluted using 0.1 % TFA in 95/5
MeCN/H2O. The product band was then eluted using 0.1 % TFA
in 50/50 MeCN/H2O. The product fraction was concentrated
under a nitrogen stream and the conjugate precipitated by the
addition of solid ammonium hexafluorophosphate. The
precipitated solids were filtered and dried to afford the
purified Ru-peptide conjugate. Yield = orange sticky solid. HPLC
(PDAD, 450 nm): Indicative Ru(II) purity vs precursors: 100 %.
1H NMR (600 MHz, CD3OD) δ (ppm): 9.74 (d, 2H); 9.06 (d, 2 H);
8.97 (m, 2 H); 8.49 (m, 2 H); 8.42 (m, 1 H); 8.33 (d, 1 H); 8.22
Synthesis of the Ru(II) conjugates
[Ru(dppz)(bpy-Ar-PEG)(bpyArCOOEt)](PF6)2 (7): A suspension of
DIPEA (15 μL, 0.086 mmol), HBTU (3.5 mg, 0.009 mmol) and (6)
(10 mg, 0.008 mmol) in 2 mL dichloromethane was allowed to
stir for 15 minutes at room temperature. To this was added a
solution of m-dPEG15-amine (7 mg, 0.010 mmol) in
dichloromethane (2 mL). The mixture was left to stir for 16
hours and was then concentrated to dryness under a nitrogen
stream. The residue was purified by column chromatography
on silica using 9/1 CH2Cl2/MeOH as eluent. The product (td, 1 H); 7.95 – 8.16 (m, 15 H); 7.91 (m, 3 H); 7.70 (m, 1 H);
7.60 (m, 1 H); 7.41 (m, 1 H); 5.34 (t, 1 H); 3.85 – 4.65 (m, 5 H);
3.62 – 3.76 (m, 16 H); 3.42 – 3.62 (m, 67 H; PEG-H); 3.37 (s, 1
H); 3.13 – 3.26 (m, 5 H); 2.98 (m, 15 H); 2.88 (s, 1 H); 2.67 (s, 1
H); 2.63 (s, 1 H); 2.24 – 2.54 (m, 3 H); 2.17 - 2.21 (m, 5 H); 1.85
– 2.23 (m, 9 H); 1.50 - 1.83 (m, 8 H); 1.44 (m, 2 H); 1.27 – 1.39
(m, 28 H); 1.25 (s, 6 H); 1.03 (dd, 2 H); 0.87 - 0.95 (m, 6 H). HR-
fraction was evaporated to dryness under nitrogen to provide
the product after acetone/diethyl ether reprecipitation as a
sticky red solid. Yield: isomer mixture, red tacky solid, 11 mg
(0.006 mmol, 71 %). HPLC (PDAD, 450 nm): Indicative Ru(II)
purity vs parent: 99.2 %. 1H NMR (600 MHz, (CD3)2CO) δ (ppm):
9.80 (m, 2 H); 9.25 (m, 2 H); 9.15 (m, 2 H); 8.69 (m, 1 H); 8.59
(m, 1 H); 8.52 (m, 2 H); 8.32 (m, 2 H); 8.09 – 8.26 (m, 13 H);
7.98 – 8.08 (m, 4 H); 7.78 (m, 1 H); 7.69 (m, 1 H); 7.46 (m, 1 H);
4.37 (2x q, 2 H, OEt -CH2-); 3.34 – 3.72 (m, 60 H, PEG -OCH2-);
3.25 (s, 3 H, PEG –OCH3); 1.37 (2x t, 3 H, OEt –CH3). HR-MS
(MALDI-QTOF) m/z: Calculated for C85H101N9O18PF6Ru [M -
PF6]+: 1782.5950; Found: 1782.6034.
MS (MALDI-QTOF) m/z: Calculated for C83H98N10O17PF6Ru
- +
[Ru(dppz)(bpy-PEG)(bpyArCONH2)
+ PF6 ] (loss of Ahx-
peptide): 1753.5791; Found: 1753.5808. Calculated for
C83H97N10O17Ru [Ru(dppz)(bpy-PEG)(bpyArCONH2) - H+]+ (loss
of Ahx-peptide): 1607.6066; Found: 1607.6180.
Photophysical characterisation
UV-Vis spectra were recorded on a Jasco spectrophotometer.
All analyses were performed using quartz cuvettes and
background correction was applied prior to measurement.
Emission spectra were recorded on a Varian Cary Eclipse
fluorescence spectrophotometer with excitation and emission
slit widths of 10 nm. Luminescent lifetimes were obtained
using a time correlated single photon counting (TCSPC)
PicoQuant system and an exciting 450 nm laser. Lifetime decay
plots were analysed using PicoQuant NanoHarp software
applying tailfit criteria; 0.9 < χ2 < 1.10. All photophysical
measurements were performed in triplicate at room
temperature (293 K).
[Ru(dppz)(bpy-Ar-PEG)(bpyArCOOH)](PF6)2 (8): The Ru-PEG
precursor (7) (10 mg, 0.005 mmol) was dissolved in 1.25 mL of
a
4/1 THF/methanol mixture under stirring at room
temperature. To this was added an aqueous solution of
LiOH.H2O (1 mg in 0.25 mL). After 2 h, the mixture was
concentrated under nitrogen to ca. 0.5 mL and treated with
0.5 mL of 0.1 M HCl and 0.5 mL of saturated aqueous
ammonium hexafluorophosphate. After diluting with 1 mL
water, the product was extracted into
4 x 2 mL
dichloromethane and the combined organic phase was washed
with 5 mL water. The separated organic layer was dried over
anhydrous magnesium sulphate, filtered and evaporated to
dryness under nitrogen. Yield = red solid, 9 mg (0.005 mmol,
91 %). HPLC (PDAD, 450 nm): Indicative Ru(II) purity vs
precursors: 99.7 %. 1H NMR (400 MHz, (CD3)2CO) δ (ppm): 9.80
(d, 2 H); 9.25 (tm, 2 H); 9.16 (m, 2 H); 8.70 (m, 1 H); 8.62 (m, 1
H); 8.53 (m, 2 H); 8.26 – 8.41 (m, 3 H); 8.08 – 8.26 (m, 11 H);
7.96 – 8.08 (m, 4 H); 7.87 – 7.96 (m, 1 H); 7.78 (m, 1 H); 7.70
(m, 1 H); 7.46 (m, 1 H); 3.38 – 3.80 (m, 60 H, PEG –OCH2-); 3.28
Conclusions
An efficient, high yielding and versatile 6 step route to tris-
heteroleptic polypyridyl Ru(II) complexes was described.
Synthesis proceeds from commercially available RuCl3, through
the [Ru(DMSO4)Cl2] precursor through stepwise coordination
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ligands in which multiple dissimilar conjugations could be
achieved. This was demonstrated by inclusion of ligands
within the tris-heteroleptic complex that contained both acid
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for postgraduate funding and Science Foundation Ireland for
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