Use of a fluorinated probe to quantitatively monitor amino acid binding preferences of ruthenium(ii) arene complexes

Article abstract of DOI:10.1039/c8dt05159c

In order to address outstanding questions about ruthenium complexes in complex biological solutions, ¹⁹F NMR spectroscopy was used to follow the binding preferences between fluorinated Ru^II(η⁶-arene)(bipyridine) complexes and protected amino acids and glutathione. Reporting what ruthenium compounds bind to in complex environments has so far been restricted to relatively qualitative methods, such as mass spectrometry and X-ray spectroscopic methods; however, quantitative information on the species present in the solution phase cannot be inferred from these techniques. Furthermore, using ¹H NMR, in water, to distinguish and monitor a number of different complex Ru^II(η⁶-arene) adducts forming is challenging. Incorporating an NMR active heteroatom into ruthenium organometallic complexes provides a quantitative, diagnostic 'fingerprint' to track solution-phase behaviour and allow for unambiguous assignment of any given adduct. The resulting ¹⁹F NMR spectra show for the first time the varied, dynamic behaviour of organoruthenium compounds when exposed to simple biomolecules in complex mixtures. The rates of formation of the different observed species are dramatically influenced by the electronic properties at the metal, even in a closely related series of complexes in which only the electron-donating properties of the arene ligand are altered. Preference for cysteine binding is absolute: the first quantitative solution-phase evidence of such behaviour.

Full text of DOI:10.1039/c8dt05159c

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Use of a fluorinated probe to quantitatively
monitor amino acid binding preferences of
Cite this: DOI: 10.1039/c8dt05159c
ruthenium(II) arene complexes†
George S. Biggs, ‡a Michael J. O’Neill,
‡
a,b Pablo Carames Mendez,§a
a
Thomas G. Scrase,¶a Yulu Lin,a Amzar Muzani Bin-Maarof,a Andrew D. Bond,
Sally R. Boss *a and Paul D. Barker
*
a
In order to address outstanding questions about ruthenium complexes in complex biological solutions,
19F NMR spectroscopy was used to follow the binding preferences between fluorinated RuII(η6-arene)
(bipyridine) complexes and protected amino acids and glutathione. Reporting what ruthenium com-
pounds bind to in complex environments has so far been restricted to relatively qualitative methods, such
as mass spectrometry and X-ray spectroscopic methods; however, quantitative information on the
species present in the solution phase cannot be inferred from these techniques. Furthermore, using 1H
NMR, in water, to distinguish and monitor a number of different complex RuII(η6-arene) adducts forming
is challenging. Incorporating an NMR active heteroatom into ruthenium organometallic complexes pro-
vides a quantitative, diagnostic ‘fingerprint’ to track solution-phase behaviour and allow for unambiguous
assignment of any given adduct. The resulting 19F NMR spectra show for the first time the varied, dynamic
behaviour of organoruthenium compounds when exposed to simple biomolecules in complex mixtures.
The rates of formation of the different observed species are dramatically influenced by the electronic pro-
perties at the metal, even in a closely related series of complexes in which only the electron-donating
properties of the arene ligand are altered. Preference for cysteine binding is absolute: the first quantitative
solution-phase evidence of such behaviour.
Received 31st December 2018,
Accepted 13th April 2019
DOI: 10.1039/c8dt05159c
rsc.li/dalton
explore the binding preferences of simple ruthenium organo-
metallic complexes for biomolecular targets, with many of
Introduction
One motivation for using ruthenium in medicinal compounds these compounds demonstrating promising anticancer
is to introduce a drug–target interaction for which the bonding activity.4–6
energy is between that of true non-covalent intermolecular
The ruthenium(III) complexes that have entered clinical
interactions and a covalent bond.1–3 In principle, tuning the trials, NAMI-A and KP1019 have received significant attention
strength of a metal–target coordination bond could be used to with respect to their distributions within plasma and in cells.
control the dissociation rate of a drug–target interaction in a They are characterized by having a number of ligands we would
way that is orthogonal to that provided by conventional medic- class as exchangeable in aqueous solution on a timescale rele-
inal chemistry. Consequently, a raft of studies have begun to vant to cellular processes. These complexes are very promiscu-
ous in complex biological mixtures, with little understanding
of their modes of action or final cellular speciation.7–9
aUniversity of Cambridge, Chemistry Department, Lensfield Road,
Ruthenium(II) arene complexes incorporating a wide range
Cambridge CB2 1EW, UK. E-mail: pdb30@cam.ac.uk, srb39@cam.ac.uk
bFaculty of Science and Engineering School of Mathematics and Physical Sciences,
of different ligands have also been studied extensively in
this context.10–16 In comparison with Ru(III) complexes, the
University of Hull, Hull HU6 7RX, UK
Ru(II)(η6-arene) bioactive scaffold has led to increased control
†Electronic supplementary information (ESI) available: Experimental procedures
and spectral data for all new compounds, together with details of the X-ray struc- over the biomolecular targets of ruthenium complexes. RAPTA-
tures of complexes [1]–[10] and [13]. CCDC 1867661–1867671. For ESI and crys-
type complexes, [Ru(arene)(PTA)X2] preferentially bind to
proteins17,18 whereas RAED-type complexes [Ru(arene)(en)Cl]
tallographic data in CIF or other electronic format see DOI: 10.1039/c8dt05159c
‡These authors contributed equally to this project.
(en = 1,2-ethylenediamine) preferentially bind to DNA; this
§Present address: School of Chemistry, University of Leeds, Leeds LS2 9JT UK.
binding can be enhanced through extended π-systems.19,20
¶Present address: Johnson Matthey plc, 28 Cambridge Science Park, Milton
Road Cambridge CB4 0FP, UK.
Although these complexes have shown promising anticancer
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activity, they are still highly promiscuous and there is little substituents into Ru(II) arene complexes can be greatly ben-
insight into their complete cellular speciation. eficial in probing their reactivity in complex mixtures, as well
The dynamic nature of the cellular concentrations and as a means of altering activity.
accessibility of biomolecules combined with the characteristi-
Throughout this study, we have used 19F NMR to gather
cally slow ligand exchange rates associated with Ru(II) arene quantitative information, such as kinetic parameters and per-
complexes,21 makes understanding the relationship between centage formation of the species present in solution, with mass
speciation of the metal complexes and cellular response pro- spectrometry used to support the assignment of species. This
blematic. A direct read out of what the ruthenium compounds combined approach can greatly simplify observing the binding
are bound to in biological environments remains challenging preferences of Ru(II) arene complexes in biological mixtures.
and has so far been restricted to relatively qualitative methods, The purpose here, was not to develop new bioactive ruthenium
such as mass spectrometry22–24 and X-ray spectroscopic organometallic compounds, but to explore a quantitative spec-
methods.25–27
NMR spectroscopy is a quantitative, sensitive, direct repor- ciation of ruthenium organometallics in complex solutions.
ter for solution behaviour. However, using 1-dimensional 1H
Incorporating the fluorine atoms into a bipyridyl ligand,
troscopic method for quickly and accurately assigning the spe-
NMR to follow the speciation of ruthenium complexes has which is stable to dissociation, enabled at least two fluorines
proved testing for anything other than the simplest examples. in chemically similar environments to be introduced to
The large number of observable proton signals together with each complex. We synthesised four fluorinated 2,2′-bipyridyl
their chemical similarity and hence proximity in the spectra derivatives; 3,3′,5,5′,6,6′-difluoro and 5,5′-di(trifluoromethyl).
severely restricts the usefulness of 1H NMR in this context. To counteract the electron withdrawing effect of the fluori-
2-dimensional NMR experiments can overcome this complex- nated ligands on the metal, we also varied the arene ligand,
ity; however, these experiments are often time consuming, including more electron-donating substituted arenes by way of
with high sample concentration necessary, if temporal resolu- compensation. The full set of complexes reported is listed in
tion is required. 31P NMR has been used extensively for follow- Table 1, and Fig. S1.†
ing the metabolic state of phosphate esters in vivo.28–30 15N
and 13C are commonly used to examine the structure and metal coordination environment and dispersion of signals
dynamics of proteins in vivo.31
that have allowed us to resolve the surprising number of
Some of these complexes provide excellent sensitivity to the
19F NMR can also be used as a sensitive probe of specific different species present. The sensitivity we report suggests
metabolic signals when a fluorine atom can be incorporated that this approach could be developed for use in vivo and we
into a small or large biomolecule as a reporter.32,33 The use of have made attempts to test that. We also show that the rates of
ligand-based fluorine NMR screening methods to rapidly formation of different species are dramatically influenced by
identify biologically active fluorinated organic compounds has the electronic properties at the metal, even in a closely related
demonstrated the usefulness of being able to detect a 19F NMR series of complexes in which only the electron-donating pro-
signal in complex environments.34,35
perties of the arene ligand are altered.
Furthermore, incorporating fluorine atoms into lead drug
compounds, has proven to be a popular way to improve the
pharmacological properties of organic-based drugs, with
approximately 20% of all currently available drugs on the
Results and discussion
market containing at least one fluorine atom.34 Recently, a flu- A number of literature preparations are available for the prepa-
oride ligand has been incorporated into the axial position of a ration of functionalised bipyridines,37,38 however, after achiev-
Pt(IV) anticancer pro-drug to enhance stability and cyto- ing relatively poor yields using copper catalysed, Ullman-type
toxicity.36 Therefore, we believe that incorporating fluorine coupling reactions, the fluorinated bipyridines were syn-
Table 1 Complexes synthesized and studied in this work, isolated and used as the hexafluorophosphate salts
Complex
Ar–X
Bipyridyl substituents R
1
2
3
4
5
6
7
8
Benzene
Toluene
p-Cymene
Hexamethylbenzene
Benzene
5,5′-Difluoro
5,5′-Difluoro
5,5′-Difluoro
5,5′-Difluoro
3,3′-Difluoro
3,3′-Difluoro
3,3′-Difluoro
3,3′-Difluoro
6,6′-Difluoro
5,5′-Di(trifluoromethyl)
None (all H)
None (all H)
Toluene
p-Cymene
Hexamethylbenzene
Benzene
Benzene
Benzene
9
10
11
12
p-Cymene
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thesised using an adapted palladium-catalysed homocoupling nucleophiles are present, but these small differences cannot
procedure in poly(ethylene) glycol (see Experimental be used to inform solution based behavior.
section).39 We found that all except the 4,4′-difluorobipyridyl
Speciation in aqueous solutions
derivative could be made reliably in good yield; we have not
pursued the 4,4′ derivative any further. The syntheses of ruthe- Before exposing the ruthenium complexes to biomolecules,
nium complexes [1]–[12] were achieved by reacting the appro- they were monitored in deuterated phosphate buffer, pD = 7.2
priate ruthenium arene dimer with a stoichiometric amount of and 1% DMF which leads to an equilibrium between chloro,
the chosen bipyridyl derivative. These complexes were charac- aquo and phosphate adducts, Fig. 1a. In order to be able to
terised by NMR, ESI-MS, elemental analysis and X-ray see as many relevant species as possible, high concentrations
diffraction.
of the complexes under study were needed so a small amount
Single crystals suitable for X-ray diffraction for complexes of DMF was used, to enhance solubility. We have seen no evi-
[1]–[10] were grown via slow diffusion of diethyl ether into a dence of DMF coordinating under these conditions. For the
saturated solution of the complex in acetone. The X-ray crystal- fluorinated complexes [1]–[8] and [10], three singlet peaks are
lographic data for all complexes are given in Tables S2–4.† The observed in the 19F NMR spectra, as represented in Fig. 1b.
novel complexes [1]–[10] adopt a pseudo-octahedral piano The behaviour of complex [9], the 6,6′-bipyridyl derivative, was
stool configuration. A representative analysis of key bond significantly different suggesting that the proximity of the flu-
lengths and angles for the 5,5′-fluorinated bipyridine series is orines to the metal impacted upon the ligand exchange pro-
1
summarised in Table S1.† Complexes [1] and [2] have shorter perties. For [11] and [12], H NMR spectra were used to resolve
Ru–Cl bond lengths than complexes [3] and [4] which could the speciation through the diagnostic metal-coordinated arene
provide a hint towards the lability of the chloride when other signals.40
Fig. 1 (a) The equilibria that exist when [Ru(η6-arene)(5,5’-difluorobipyridine)]+ complexes are incubated in phosphate buffer. (b) Time course 19F
{1H} NMR spectra of complex [4] incubated in 10 mM deuterated phosphate buffer (2 mM Ru, pD = 7.2, 310 K). After 24 h, 1 eq. of AgNO3 was added
to abstract the chloride ligand and encourage formation of the aquo complex. (c) 19F{1H} NMR spectra of complex [2] incubated in D2O and buffered
D2O of different phosphate concentration (2 mM Ru, pD = 7.2 (when buffered), 2 h, 310 K). (d) Mass spectra recorded of the solution mixture when
complex [2] is incubated in D2O and buffered D2O, expected masses quoted in Table S6.†
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With a number of titratable groups involved in these [10] and [11]) and for the 5,5-difluorobipyridyl complex with
systems, examining the ligand exchange behaviour of these different arene ligands (complexes [1]–[4]). The observed differ-
complexes around conditions of pH relevant to biological con- ences in pKa are entirely consistent with the changes in elec-
ditions requires strong buffering. We chose phosphate as a tron density at the metal due to the subtleties of the electron
buffer for its relevance to biological conditions. However, it is withdrawing effects of fluorines on the bipyridyl ligand and
clear that phosphate competes quite strongly as a ligand to the the electron donating effects of the different arenes.42 In other
metal, Fig. 1c, and the presence of phosphate species have words, the trend observed going from complex [1]–[4] shows
been confirmed by mass spectrometry, Fig. 1d and Table S6.† that the increasing electron donating capability of the η6-arene
Sadler et al. showed that the structurally related [Ru(η6-arene) ligand to the ruthenium centre leads to a higher measured pKa
(en)Cl]+ initially binds to the phosphate in the nucleobase value; an increased electron density on the metal lowers its
5-GMP before being displaced by the guanine N7.41 To main- Lewis acidity. The electron withdrawing capabilities of the tri-
tain a balance between buffering strength and introducing a fluoromethyl groups on complex [10] significantly increases
competing ligand a phosphate buffer concentration of 10 mM the Lewis acidity, reflected in the lowest pKa measured for any
was chosen for all incubations, when the ruthenium concen- of these complexes. Interestingly, this pKa had to be measured
1
tration was 2 mM.
using H spectra because the 19F resonances in this complex,
19F resonances from the aquo and phosphate species are where the fluorine atoms are not directly on the bipyridyl ring,
pH dependent and therefore the pKa for the deprotonation of are not sensitive enough to changes in coordination to accu-
the bound aquo ligand can easily be measured. Fig. 2a, b and rately measure the pKa. Clearly this has implications for the
c show representative data and calculated pKa values for the general use of 19F on spectator ligands for reporting changes
benzene complexes with different bipyridyls (complexes [1], at the metal site.
Fig. 2 (a) pH dependence of the chemical shifts of the aquo coordinated ruthenium species and determination of the pKa in the following com-
plexes [1] – red circles; [2] – blue squares; [3] – pink triangles; [4] – black circles; [10] – green squares; [11] – orange triangles. 19F chemical shifts of
the 5,5’-difluorobipyridyl fluorines or 1H chemical shift of the coordinated benzene ligand were plotted against pD. The lines are a least squares fit to
an equation involving a single titratable group. (b) A series of 19F{1H} spectra of complex [1] in D2O phosphate buffer at differing pD values (2 mM Ru,
298 K). (c) The pKa values of a series of ruthenium complexes measured using (where applicable) both 19F{1H} NMR and 1H NMR. (a) Unable to
measure pKa from data collected. (b) No fluorine atoms present in complex.
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Diagnostic determination of amino acid binding
Reaction of these probes with N- and/or C-protected amino
acids gave a series of characteristic 19F NMR signals, allowing
for unambiguous assignment of any given adduct. We note
that the fluorines on the bipyridyl ring are diastereotopic and
once a chiral amino acid ligand is coordinated to a ruthenium
in the piano stool complex they are magnetically inequiva-
lent.43 Therefore, despite the symmetry of the starting mole-
cule, the amino acid coordinated adducts of these complexes
don’t always give rise to single resonances in the proton
decoupled 19F NMR spectra.
It is therefore necessary to point out how we have inter-
preted the spectra and especially how the diastereotopic
context of the fluorine atoms in the 5,5′-bipyridyl positions
results in more complex 19F NMR spectra than one might
expect. For instance, when complex [1] is incubated with
N-acetyl L-glutamine two doublets separated by 0.18 ppm are
observed, which we attribute to one species with the glutamine
coordinated via the α-carboxylate of the amino acid. That these
signals originate from the same species is clearly evidenced by
the 8JF–F coupling of 2.26 Hz observed at high resolution and a
clear cross-peak in a proton decoupled 19F–19F COSY experi-
ment (Fig. 3). When achiral amino acids (e.g. N-acetyl glycine)
are coordinated through the α-carboxylate only a singlet is
observed, as expected (Fig. S2†).
Fig. 4 A series of 19F{1H} NMR spectra of complexes [1], [3], [5] and [7]
incubated with N-acetyl cysteine methyl ester (2 mM Ru, 3 eq. amino
acid, 310 K). The chemical shift scale is the same for all spectra, which
are aligned to the chloride peak in each case. Absolute δ values given in
Table S5.†
ligand bond that could cause an environment that breaks the
symmetry (Fig. S3†).
However, the consequences of the diastereotopic fluorines
are not always manifest in the spectra. In the case of the 3,3′-
difluorobipyridyl complexes, [5] (Ar = benzene) and [7] (Ar =
p-cymene), only one Ru-cysteine 19F NMR signal is observed,
see Fig. 4. The crystal structure of the 3,3′-difluorobipyridyl
containing complex, [5], shows that the organometallic ion is
in fact chiral at the metal due to the adjacent fluorines being
forced out of plane by each other (both enantiomers are
present in the centrosymmetric crystal structure, see ESI†).
Should a chiral amino acid coordinate in place of the chloride,
this would technically generate diastereomers. Presumably the
metal–ligand coordination sphere is flexible enough in solu-
tion for there to be fluxionality between the two non-planar
pyridyl rings such that the two fluorine environments appear
equivalent and we observe only one 19F NMR signal, the non-
chiral average. So, although coordination of the 3,3′-difluorobi-
pyridine ligand to the ruthenium piano stool should also lead
to diastereotopic fluorines, they appear in the same magnetic
environment and their signals do not split into two.
Under similar conditions,
a ruthenium species with
N-acetyl cysteine methyl ester coordinated can also give more
than one peak (see Fig. 4, complexes [1] (Ar = benzene) and [3]
(Ar = p-cymene). In the case of complex [1], variable tempera-
ture experiments show that this really is two coupled doublets
with very strong roofing (Fig. S3†). The chemical shift separ-
ation between the diastereotopic fluorine signals is small and
consequently this peak often appears as one broad resonance.
Again, achiral thiolate molecules (cystamine for instance)
clearly coordinate through the sulfur and yield a sharp singlet
in the proton decoupled 19F{1H} spectrum (Fig. S2†).
Additionally, the variable temperature experiments provided
no indication that there is restricted rotation about the metal
The extra complexity in the signals observed with the fluor-
ines in the 5,5′-bipyridyl positions reduces the sensitivity of
the experiment given that we generate two 19F signals for each
amino-acid coordinated ruthenium complex rather than one
intense singlet. However, the sophistication in the level of
detail we can observe with this technique is exemplified where
the diastereotopic nature of the fluorines is apparent in the
spectra. Therefore, we pursued the 5,5′-difluorobipyridyl com-
plexes to give us the most complete understanding of the com-
plexes under discussion but noting that the 3,3′-difluorobipyri-
dine complexes might be more suited to further studies of
more complex mixtures of species.
Influence of the arene ligand on complex speciation
The 19F NMR spectra of the 5,5′-difluorobipyridyl complexes
[1]–[4] incubated with N-acetyl cysteine methyl ester, Fig. 5,
show the extent to which ligand exchange varies with different
Fig. 3
A
19F{1H}–19F{1H} COSY spectra from the incubation of complex
[1] with N-acetyl L-glutamine (2 mM Ru, 3 eq. amino acid, starting pD =
7.2, 24 h, 310 K).
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These incubations were followed by 19F{1H} and 1H NMR,
and mass spectrometry (Table S7†) over a 24-hour period at
310 K in 10 mM D2O phosphate buffer with starting pD = 7.2.
These temporal experiments enabled us to identify and quan-
tify signals due to ruthenium-amino acids adducts, e.g.
complex [1] with cysteine, Fig. S5.† The data from these incu-
bations is summarised in Table 3 for the two electronic
extremes, the benzene arene, complex [1], and the hexamethyl-
benzene arene, complex [4]. Given that the coordination of
some amino acid side chains results in a release of protons it
proved difficult to clamp the pD tightly in the experiments
described below, even with 10 mM phosphate buffer. The exact
change in pD of any experiments described below could be
measured intrinsically from the chemical shift of the 19F
signals from each specific aquo complex, which are exception-
ally sensitive to changes in pD. The greatest adduct formation
is observed for both complexes [1] and [4] with N-Ac-cysteine-
OMe. Furthermore, the adduct formation with [4] is much
greater for the softer side chains of cysteine, histidine and
methionine.
Fig. 5 19F{1H} NMR spectra of complexes [1], [2], [3] and [4] incubated
with N-acetyl cysteine methyl ester (2 mM Ru, 3 eq. amino acid, 24 h,
310 K). The chemical shift scale is the same for all spectra, which are
aligned to the chloride peak in each case. Absolute δ values given in
Table S5.†
arene ligands. In addition, the electron donating properties of
the arene ligand influence the chemical shift separation of the
doublets with the hexamethylbenzene complex, [4] providing
the greatest resolution.
The formation of the ruthenium cysteine adducts have
been measured over a short period and rate constants
measured, Table 2 and Fig. S4.† There is an initial rapid
approach to equilibrium between the chloride, aquo and phos-
phate species. The observed rate of cysteine complex formation
is adequately described by a 1st order process, therefore, we
believe ligand dissociation to be the rate determining step.
The benzene and tolyl complexes, [1] and [2] have comparable
rate constants, however, the cymene and hexamethylbenzene
complexes [3] and [4] are slightly faster, as expected, due to the
increased electron donating capabilities of the cymene and
hexamethylbenzene ligands.
A screen of complex [1] with all of the naturally-occurring
amino acids with side chains suitable for metal coordination
allowed us to identify those amino acids (cysteine, glutamic
acid, histidine and methionine) that give detectable metal
complexes at relatively low adduct concentrations. These
amino acids were protected at both the amino and carboxylate
ends, to limit binding to amino acid side chains, and incu-
bated in an excess of the ruthenium complexes [1]–[4]. The
chosen amino acids offer some side chain variety given that we
predicted that cysteine and methionine would coordinate
through sulphur, glutamic acid through oxygen and histidine
through nitrogen.
Competition experiments for amino-acid binding
The 19F{1H} NMR spectra of complex [1] after 24 hours (where
the reaction was essentially at equilibrium) when incubated
with N-protected esters of cysteine, glutamic acid, histidine
and methionine, are presented in Fig. 6 and show metal
coordination to all four, distinct amino acid side chains. The
preference for cysteine binding is validated through a compe-
tition study involving a mixture of all four amino acids which
confirms the first direct evidence for the much-asserted claim
that ruthenium(II) organometallic complexes preferably bind
to sulphur centres in a biological context. The extent of this
preference is remarkable, especially when compared to meth-
ionine, whose thioether donor group is almost completely out-
competed by cysteine’s thiolate. Surprisingly, it also outcom-
petes histidine under our conditions. The difference in the
chemical shift of the aquo species indicates that the pD after
24 hours of incubation is no longer 7.20, due to release of
protons from amino acid coordination. This change is empha-
Table 3 Reactions of complexes [1] and [4] with protected amino acids
(2 mM Ru, 1.5 eq. amino acid, buffer starting pD = 7.2, 310 K). % reaction
is calculated through integrating [Ru]-amino acid peak with respect to
the peak areas of the chloride, aquo and phosphate species. pD is
measure from the δ of the aquo species
% reaction
pD
Table 2 A comparison of the rate constants for the complexes [1]–[4]
for the formation of a ruthenium cysteine adduct (2 mM Ru, 3 eq. amino
acid, 310 K)
Species Amino acid
2 hours 24 hours 2 hours 24 hours
[1]
N-Ac-Cys-OMe
N-Z-Glu-OMe
N-Bz-His-OMe
N-Ac-Met-OMe
N-Ac-Cys-OMe
N-Z-Glu-OMe
N-Bz-His-OMe
N-Ac-Met-OMe
12.9
12.8
0.7
55.4
13.6
8.3
11.6
77.5
5.3
6.60
6.10
7.26
7.14
7.18
6.27
7.53
7.26
6.38
6.04
7.10
7.10
6.97
6.26
7.47
7.25
Complex
kobs/×10−4 s−1
0.7
[1]
[2]
[3]
[4]
2.17 0.03
2.08 0.05
2.76 0.04
3.71 0.08
[4]
30.0
4.3
1.4
23.1
45.3
7.3
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Fig. 6 A series of 19F{1H} NMR spectra when complex [1] is incubated with the protected amino acids, N-Ac-Cys-OMe, N-Z-Glu-OMe, N-Bz-His-
OMe, N-Ac-Met-OMe, and a mixture of all amino acids together, (2 mM Ru, 3 eq. each amino acid, 24 h, 310 K).
sised in these experiments due to the working pD being near thioether coordination generates a new chiral centre at the
the pKa for the ruthenium aquo species, and this does not dis- sulphur and the two consequent diastereomers are not formed
tract from the key competition experiments presented in equally. For histidine coordination the diastereotopic fluorines
Fig. 6.
described above are more obvious, but again two doublets of
In principle, of course, one could use high-resolution, 1H unequal intensity are observed and this is ascribed to species
NMR data to uncover the speciation we have demonstrated that differ by coordination through either of the imidazole
above using 19F NMR. The enhanced clarity of using 19F NMR nitrogens, Nδ or Nε.
to monitor speciation of ruthenium complexes in complex bio-
We have tried to crystallise the amino acid-coordinated
logical mixtures is demonstrated by comparing the time complexes in many instances, but the only example that has
course 19F NMR and 1H NMR spectra when complex [1] is incu-
bated with the four protected amino acids in competition as
discussed above, Fig. S6.† The 19F NMR spectra allow for rapid
(32 scans, 2 minutes measurement) identification of the
number and concentration of different ruthenium species in
1
solution. The H NMR spectra are, however, difficult to decon-
volute without time consuming 2-D NMR experiments, and
peaks often overlap or are co-incident. The data in Fig. S6† are
for the best-case scenario; for complexes containing any arene
ligand other than benzene, 1H NMR data were insufficient,
making accurate quantification with temporal resolution
impossible.
It is worth explaining here that the signals observed from
methionine, glutamic acid and histidine coordinated species
reveal additional layers of complexity leading to multiple reso-
nances in the 19F NMR spectra. While the methionine species
in Fig. 6 generates what looks like a singlet, binding to com-
Fig. 7 Structure of complex [13] showing displacement ellipsoids at
plexes [2]–[4] gives two doublets of unequal intensity
50% probability. The counterions, hydrogen atoms and lattice water
(Fig. S7–9†). Our interpretation of this is that methionine molecules have been omitted for clarity.
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Fig. 8 A series of 19F{1H} NMR spectra when complex [1] is incubated with defined mixtures of reduced and oxidised glutathione (2 mM Ru, 3 eq.
glutathione, 24 h, 310 K).
yielded diffraction quality crystals is that of [Ru(η6-benzene) tathione. The cell redox buffer, glutathione, equilibrates
(2,2′-bipyridine)(N-acetyl cysteine)][PF6], [13], synthesised from between reduced monomer (GSH, containing a thiol) and oxi-
[11] in an excess of cysteine (Fig. 7). There is nothing surpris- dized dimer (GSSG, containing a S–S bridge).
ing about this particular structure in terms of bond lengths
Incubating [1] with varying defined mixtures of GSH and
and angles. The crystal lattice is stabilised primarily by GSSG gives 19F NMR spectra which quantifiably show that
H-bonding between the amino acid NH and CO groups and binding to the acid groups is preferred in the absence of free
water, together with stacking of the bipyridyl rings.
thiolate, but that binding to cysteine’s thiolate group is mark-
When incubated with N-acetyl cysteine, the ruthenium edly thermodynamically preferred in the regimes for which it
complex is exposed to nucleophilic attack from both the α car- is available (Fig. 8). Complexes [2], [3] and [4] also exhibit pre-
boxylate and thiolate Lewis bases. Interestingly, temporal ferential thiolate coordination (Fig. S11–13†).
studies of these samples, show that the carboxylate co-
ordinated species form most rapidly, before being replaced by
the thiol coordinated species (Fig. S10†). The crystal structure
reports the preference for metal-thiolate adduct formation in
the solid state. The NMR spectroscopy and crystallographic evi-
Conclusions
19F NMR spectroscopy has enabled us to undertake a detailed
study of a suite of ruthenium organometallic complexes with
fluoro-substituted bipyridyl ligands and their ligand exchange
behaviour with amino acid side chains. The 19F NMR spectra
provide quantitative information on the reactions of these
complexes with amino acid Lewis bases which couldn’t be
dence suggest that these ruthenium(II) arene bipyridine com-
plexes form surprisingly stable cysteine adducts in contrast to
other structurally similar complexes whereby cysteine coordi-
nation appears to catalyse further decomposition.44–46
1
gathered using traditional 1-D H NMR (2-D experiments rely
on extensive measurement time). Specifically, we can assess
Binding to glutathione
Reasoning that complex [1] would bind to cysteine in a cell if the relative binding preferences for each metal complex and
presented with the opportunity, we probed its reactivity with track the speciation of the complexes on a relevant time scale.
the most bioavailable source of cellular cysteine residues: glu- While our question is ultimately to ask whether these or
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related complexes might report what they are bound to in Syntheses
complex biological mixtures, perhaps even cellular cytoplasm,
the complexity of signals even in relatively simple mixtures at The detailed syntheses and characterisation of the ruthenium
moderate concentrations surprised us and this required arene dimers, fluorinated bipyridine ligands (A)–(D) and ruthe-
careful and detailed analysis to ensure that the output from nium complexes [1]–[10] are presented in the ESI.†
the 19F NMR could be accurately interpreted.
Representative syntheses of the fluorinated bipyridines and
Of course, the main targets for ruthenium organometallics ruthenium bipyridine complexes are as follows:
in cells are likely to be proteins which can provide one or more
General procedure for the synthesis of fluorinated bipyri-
Lewis basic sidechains at a given site to exchange with the dine ligands (a)–(d). The starting bromopyridine (1 mol
metal. We will extend our 19F reporter studies to examine spe- equiv.), PdII(OAc)2 (0.025 mol equiv.), K2CO3 (1 mol equiv.)
ciation following reaction with proteins, but early experiments and poly(ethylene glycol) (Mw 4000, 5.0–10 g) were combined
show that this cannot be generalised for all proteins and in a nitrogen purged flask. This mixture was heated to 120 °C
results are, unsurprisingly, very protein specific. However, we and the temperature maintained for 48 hours with stirring.
believe the insight provided by the experiments described The mixture was cooled to 80 °C and 15 mL of warm water was
above are not just useful, but essential to understand specia- added. Once at room temperature a further 10 mL of water was
tion of ruthenium complexes in complex biochemical added and the suspension exhaustively extracted with ethyl
environments.
acetate. The combined extracts were washed with saturated
aqueous Na2S2O3 and three times with brine. The organic layer
was then dried over MgSO4 and solvent removed in vacuo. The
product was then purified via sublimation.
Experimental section
General considerations
General procedure for the synthesis of Ru complexes.
[RuCl2(η6-arene)]2 (1 mol equiv.) and (a), (b), (c) or (d) (2 mol
equiv.) were added to a nitrogen purged flask. Freshly distilled
MeOH was added and the reaction was stirred for 24 hours at
room temperature. The contents were filtered under gravity to
remove unreacted ruthenium dimer and the solution was
reduced to approximately 5 mL in vacuo. NH4PF6 (6 mol
equiv.) was added and the mixture was shaken and left at
−10 °C for a further 24 hours. The product was collected by
gravity filtration as an orange/yellow solid and washed with
cold MeOH and Et2O. Purification could be achieved through
recrystallization from hot MeOH.
When necessary all reactions were kept under an inert atmo-
sphere or under a N2 flow using standard Schlenk line tech-
niques. Methanol (MeOH), acetonitrile (MeCN), dichloro-
methane (DCM) and other required solvents were dried
through distillation, stored over suitable drying agents and
purged with N2 before use.
Chemicals and reagents
Chemicals and solvents were purchased from chemical suppli-
ers Sigma-Aldrich, Merck, Acros Organic and Insight
Biotechnologies.
Synthesis of [13]-[Ru(η6-benzene)(2,2′-bipyridine)(N-acetyl
cysteine)][PF6]. In
a typical experiment N-acetyl-cysteine
(21 mg, 0.12 mmol) was dissolved in water (8 mL) and
complex [8] (30 mg, 0.058 mmol) added forming a yellow solu-
Physical measurements
Mass spectrometry was performed on a Micromass Quattro LC tion that turned orange after 18 hours. The reaction mixture
ESI-mass spectrometer. Samples were typically prepared in was stirred at 60 °C for 72 hours before being cooled to RT and
ultrapure water or 50 : 50 ultrapure water/acetonitrile. then stored at 4 °C for 72 hours. The resulting red crystals,
Ionization was achieved with a capillary voltage of 2.8 kV, cone suitable for single crystal X-ray diffraction, were collected on
voltage of 30 V and a collection voltage of 3 V. Desolvation and the filter and washed with ice cold water (1 mL) before being
capillary temperatures were 313 K.
dried in vacuo.
1H NMR: (400.13 MHz, d6-DMSO): 9.09 (dd, JHH = 11.0 Hz,
3
Single-crystal X-ray diffraction data were collected at 180 K
using either a Nonius KappaCCD or Bruker D8-QUEST diffract- 3JHH = 5.4 Hz, 2H, 6,6′-position), 8.33 (dd, 3JHH = 8.2 Hz, 3JHH
=
ometer, equipped with MoKα or CuKα radiation, respectively. 6.0 Hz, 2H, 3,3′-position), 8.09 (overlapping ddd, 3JHH = 8.2 Hz,
Structures were solved using SHELXT47 and refined on F2 3JHH = 8.0 Hz, JHH = 5.4 Hz, 2H, 4,4′-position), 7.79 (overlap-
4
using SHELXL.48
ping ddd, 3JHH = 11.0 Hz, 3JHH = 8.0 Hz, 3JHH = 6.0 Hz, 2H, 5,5′-
1H, 13C, 19F and two-dimensional NMR spectra were col- position), 5.95 (s, 6H, PhH), 3.74 (m; 1H N-Ac-Cys-C*H
lected using a 400 MHz Bruker Avance III. 1H NMR spectra (m; 2H; N-Ac-Cys-CH ). 13C{1H}
2), 1.75 (s; 3H; N-Ac-Cys-CH3
were collected at 400.13 MHz, 13C NMR spectra at 100.57 MHz NMR: (100.57 MHz, D2O): 174.2, 173.3 (N-Ac-Cys-CH3C
ONH- +
and 19F 376.50. All 1D 19F NMR spectra were recorded using an N-Ac-Cys-C
OOH), 155.2 (6,6′-position, 154.7 (2,2′-position),
inverse gated decoupling pulse programme so that integration 139.5 (4,4′-position) 127.2 (5,5′-position), 123. (3,3′-Position),
values can be quantified. Fluorine COSY experiments were col- 88.5 (PhH), 54.7 (N-Ac-Cys-C*H), 29.2 (N-Ac-Cys-CH2), 21.4
lected using a 400 MHz Bruker QNP Cryoprobe spectrometer. (N-Ac-Cys-CH3,). IR: ν(N–H) 3648 (m); (X–H) multiple weak
Elemental (C, H, N) analyses were obtained using an Exeter signals around 3000; (CvO acid) 1735 (m); (CvO amide) 1607
̲,), 2.12
̲
̲
̲
̲
̲
̲
̲
CE-440 Elemental Analyser.
(s); (arene stretches) 1471 (m) & 1445 (s); (arene bends) 837 (s),
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820 (s), 763 (s) & 726 (m). LR-MS: (ESI+ m/z 498.0 (singly
charged, [Ru(η6-benzene)(2,2′-bipyridine)(N-acetyl-cysteine)]+).
2 M. J. Clarke, Coord. Chem. Rev., 2002, 232, 69–93.
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pKa Measurements
4 C. Artner, H. U. Holtkamp, C. G. Hartinger and
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327.
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10 mM deuterated phosphate buffer solution was prepared
using potassium dihydrogen orthophosphate in D2O and
adjusted to different pD values in the range of 4.7 to 11.2
using 1 N NaOD. The different buffers were then incubated
with complexes [1]–[4], [10] and [11] for 2 hours at 298 K and
1H and 19F{1H} NMR spectra recorded. All 19F{1H} NMR
spectra recorded are 32 scans. pD values are corrected pH
meter readings.
7 M. Groessl, E. Reisner, C. G. Hartinger, R. Eichinger,
O. Semenova, A. R. Timerbaev, M. A. Jakupec, V. B. Arion
and B. K. Keppler, J. Med. Chem., 2007, 50, 2185–2193.
8 R. Trondl, P. Heffeter, C. R. Kowol, M. A. Jakupec,
W. Berger, B. K. Keppler, J. C. Bendell, A. Ogden, D. D. Von
Hoff, U. Jaehde, R. Stoika, A. Zaichenko and W. Berger,
Chem. Sci., 2014, 5, 2925–2932.
Biomolecule interactions
The incubations between ruthenium and amino acids were
performed in either a 1.5 or 3 molar excess of amino acid/glu-
tathione. Ruthenium complexes were dissolved in 1% DMF
and diluted with a solution of amino acid in D2O phosphate
buffer pD = 7.2. 19F{1H} NMR spectra were recorded at 2, 4, 8
and 24 hours, with mass spectra recorded at 8 and 24 hours.
All 19F{1H} NMR spectra recorded are 32 scans.
9 M. Groessl, O. Zava and P. J. Dyson, Metallomics, 2011, 3,
591–599.
10 S. M. Meier-Menches, C. Gerner, W. Berger, C. G. Hartinger
and B. K. Keppler, Chem. Soc. Rev., 2018, 47, 909–928.
11 P. Zhang and P. J. Sadler, J. Organomet. Chem., 2017, 839,
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Accession Codes
CCDC 1867661–1867671 contain the supplementary crystallo-
graphic data for this paper.
12 A. A. Nazarov, C. G. Hartinger and P. J. Dyson,
J. Organomet. Chem., 2014, 751, 251–260.
13 A. L. Noffke, A. Habtemariam, A. M. Pizarro and
P. J. Sadler, Chem. Commun., 2012, 48, 5219–5246.
14 M. Kubanik, W. Kandioller, K. Kim, R. F. Anderson,
E. Klapproth, M. A. Jakupec, A. Roller, T. Söhnel,
B. K. Keppler and C. G. Hartinger, Dalton Trans., 2016, 45,
13091–13103.
15 T. G. Scrase, M. J. O’Neill, A. J. Peel, P. W. Senior,
P. D. Matthews, H. Shi, S. R. Boss and P. D. Barker, Inorg.
Chem., 2015, 54, 3118–3124.
16 J. M. Cross, N. Gallagher, J. H. Gill, M. Jain,
A. W. McNeillis, K. L. Rockley, F. H. Tscherny,
N. J. Wirszycz, D. S. Yufit and J. W. Walton, Dalton Trans.,
2016, 45, 12807–12813.
Author contributions
GSB and MJO contributed equally to this project. MJO had the
original idea; MJO, GSB, TGS, YL, SRB and PDB designed
experiments; GSB, MJO, TGS, PCM, AMBM, and YL carried out
experimental work; ADB collected X-ray diffraction data and
solved the structures; all authors analysed experimental data;
GSB, MJO, PDB and SRB wrote the manuscript.
Conflicts of interest
17 M. V. Babak, S. M. Meier, K. V. M. Huber, J. Reynisson,
A. A. Legin, M. A. Jakupec, A. Roller, A. Stukalov,
M. Gridling, K. L. Bennett, J. Colinge, W. Berger,
P. J. Dyson, G. Superti-Furga, B. K. Keppler and
C. G. Hartinger, Chem. Sci., 2015, 6, 2449–2456.
18 B. Wu, M. S. Ong, M. Groessl, Z. Adhireksan,
C. G. Hartinger, P. J. Dyson and C. A. Davey, Chem. – Eur. J.,
2011, 17, 3562–3566.
19 Z. Adhireksan, G. E. Davey, P. Campomanes, M. Groessl,
C. M. Clavel, H. Yu, A. A. Nazarov, C. H. F. Yeo, W. H. Ang,
P. Dröge, U. Rothlisberger, P. J. Dyson and C. A. Davey, Nat.
Commun., 2014, 5, 1387–1407.
The authors declare no competing financial interest.
Acknowledgements
TGS and MJO were supported by EPSRC studentships (EP/
P505445/1 and EP/K503009/1) and GSB was supported by the
EPRSC (EP/N509620/1) and Peterhouse, Cambridge. We thank
the staff of the Chemistry Department NMR facility for help
and advice.
20 Z. Ma, G. Palermo, Z. Adhireksan, B. S. Murray, T. von
Erlach, P. J. Dyson, U. Rothlisberger and C. A. Davey,
Angew. Chem., Int. Ed., 2016, 55, 7441–7444.
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