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Journal ArticleDOI

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

21 May 2019-Dalton Transactions (The Royal Society of Chemistry)-Vol. 48, Iss: 20, pp 6910-6920

TL;DR: 19F NMR spectroscopy was used to follow the binding preferences between fluorinated RuII(η6-arene)(bipyridine) complexes and protected amino acids and glutathione, showing for the first time the varied, dynamic behaviour of organoruthenium compounds when exposed to simple biomolecules in complex mixtures.

AbstractIn 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 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 RuII(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.

Topics: Fluorine-19 NMR (57%), Amino acid binding (56%), Ruthenium (56%), Proton NMR (51%), Bipyridine (51%)

Summary (2 min read)

INTRODUCTION:

  • One motivation for using ruthenium in medicinal compounds is to introduce a drug-target interaction for which the bonding energy is between that of true non-covalent intermolecular interactions and a covalent bond.
  • 10–16 In comparison with RuIII complexes, the RuII(h6-arene) bioactive scaffold has led to increased control over the biomolecular targets of ruthenium complexes.
  • 19,20 Although these complexes have shown promising anticancer activity, they are still highly promiscuous and there is little insight into their complete cellular speciation.
  • Incorporating the fluorine atoms into a bipyridyl ligand, which is stable to dissociation, enabled at least two fluorines in chemically similar environments to be introduced to each complex.

RESULTS AND DISCUSSION

  • A number of literature preparations are available for the preparation of functionalised bipyridines,37,38 however, after achieving relatively poor yields using copper catalysed, Ullman-type coupling reactions, the fluorinated bipyridines were synthesised using an adapted palladium-catalysed homocoupling procedure in poly glycol (see Experimental Section).
  • The authors found that all except the 4,4’-difluorobipyridyl derivative could be made reliably in good yield; they have not pursued the 4,4’ derivative any further.
  • The synthesis of the ruthenium complexes [1] – [12] was achieved by reacting the appropriate ruthenium arene dimer with a stoichiometric amount of the chosen bipyridyl derivative.
  • These complexes were characterised by NMR, ESI-MS, elemental analysis and X-ray diffraction.
  • Complexes [1] and [2] have shorter Ru-Cl bond lengths than complexes [3] and [4] which could provide a hint towards the lability of the chloride when other nucleophiles are present, but these small differences cannot be used to inform solution based behavior.

Speciation in Aqueous Solutions

  • Before exposing the ruthenium complexes to biomolecules, they were monitored in deuterated phosphate buffer, pD = 7.2 and 1% DMF which leads to an equilibrium between chloro, aquo and phosphate adducts, Figure 1a.
  • It is therefore necessary to point out how the authors have interpreted 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.
  • The enhanced clarity of using 19F NMR to monitor speciation of ruthenium complexes in complex biological mixtures is demonstrated by comparing the time course 19F NMR and 1H NMR spectra when complex [1] is incubated with the four protected amino acids in competition as discussed above, Figure S6.
  • The crystal lattice is stabilised primarily by H-bonding between the amino acid NH and CO groups and water, together with stacking of the bipyridyl rings.

Binding to Glutathione

  • Reasoning that complex [1] would bind to cysteine in a cell if presented with the opportunity, the authors probed its reactivity with the most bioavailable source of cellular cysteine residues: glutathione.
  • The cell redox buffer, glutathione, equilibrates between reduced monomer (GSH, containing a thiol) and oxidized dimer (GSSG, containing a S-S bridge).
  • Incubating [1] with varying defined mixtures of GSH and GSSG gives 19F NMR spectra which quantifiably show that binding to the acid groups is preferred in the absence of free thiolate, but that binding to cysteine’s thiolate group is markedly thermodynamically preferred in the regimes for which it is available .
  • Complexes [2], [3] and [4] also exhibit preferential thiolate coordination .

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 gathered using traditional 1-D 1H NMR (2-D experiments rely on extensive measurement time).
  • Specifically, the authors can assess the relative binding preferences for each metal complex and track the speciation of the complexes on a relevant time scale.
  • Of course, the main targets for ruthenium organometallics in cells are likely to be proteins which can provide one or more Lewis basic sidechains at a given site to exchange with the metal.
  • The authors will extend their 19F reporter studies to examine speciation following reaction with proteins, but early experiments show that this cannot be generalised for all proteins and results are, unsurprisingly, very protein specific.

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1
Use of a Fluorinated Probe to Quantitatively Monitor Amino Acid
Binding Preferences of Ruthenium(II) Arene Complexes
George S. Biggs,
Michael J. O’Neill,
†,
Pablo Carames Mendez, Thomas G. Scrase, Yulu
Lin, Amzar Muzani Bin-Maarof, Andrew D. Bond, Sally R. Boss* and Paul D. Barker*
University of Cambridge, Chemistry Department, Lensfield Road, Cambridge CB2 1EW,
UK
Faculty of Science and Engineering School of Mathematics and Physical Sciences,
University of Hull, Hull, UK
These authors contributed equally to this project.
ABSTRACT:
In order to address outstanding questions about ruthenium complexes in complex
biological solutions,
19
F NMR spectroscopy was used to follow the binding preferences
between fluorinated Ru
II
(h
6
-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
1
H NMR,
in water, to distinguish and monitor a number of different complex Ru
II
(h
6
-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
19
F 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.
INTRODUCTION:
One motivation for using ruthenium in medicinal compounds is to introduce a drug-target
interaction for which the bonding energy is between that of true non-covalent
intermolecular interactions and a covalent bond.
1–3
In principle, tuning the strength of a
metal-target coordination bond could be used to control the dissociation rate of a drug-
target interaction in a way that is orthogonal to that provided by conventional medicinal
chemistry. Consequently, a raft of studies have begun to explore the binding preferences
of simple ruthenium organometallic complexes for biomolecular targets, with many of
these compounds demonstrating promising anticancer activity.
4–6
The ruthenium complexes that have entered clinical trials, NAMI-A and KP1019 have
received significant attention with respect to their distributions within plasma and in cells.
They are characterized by having a number of ligands we would class as exchangeable in
aqueous solution on a timescale relevant to cellular processes. These complexes are very
promiscuous in complex biological mixtures, with little understanding of their modes of
action or final cellular speciation.
7–9

2
Ruthenium(II) arene complexes incorporating a wide range of different ligands have also
been studied extensively in this context.
1016
In comparison with Ru
III
complexes, the
Ru
II
(h
6
-arene) bioactive scaffold has led to increased control over the biomolecular targets
of ruthenium complexes. RAPTA-type complexes, [Ru(arene)(PTA)X
2
] preferentially bind
to proteins
17,18
whereas RAED-type complexes [Ru(arene)(en)Cl] (en = 1,2-
ethylenediamine) preferentially bind to DNA; this binding can be enhanced through
extended p-systems.
19,20
Although these complexes have shown promising anticancer
activity, they are still highly promiscuous and there is little insight into their complete
cellular speciation.
The dynamic nature of the cellular concentrations and accessibility of biomolecules
combined with the characteristically slow ligand exchange rates associated with Ru(II)
arene complexes,
21
makes understanding the relationship between speciation of the metal
complexes and cellular response problematic. A direct read out of what the ruthenium
compounds are bound to in biological environments remains challenging and has so far
been restricted to relatively qualitative methods, such as mass spectrometry
2224
and X-ray
spectroscopic methods.
2527
NMR spectroscopy is a quantitative, sensitive, direct reporter for solution behaviour.
However, using 1-Dimensional
1
H NMR to follow the speciation of ruthenium complexes
has proved testing for anything other than the simplest examples. The large number of
observable proton signals together with their chemical similarity and hence proximity in the
spectra severely restricts the usefulness of
1
H NMR in this context. 2-Dimensional NMR
experiments can overcome this complexity; however, these experiments are often time
consuming, with high sample concentration necessary, if temporal resolution is required.
31
P NMR has been used extensively for following the metabolic state of phosphate esters
in vivo.
2830
15
N and
13
C are commonly used to examine the structure and dynamics of
proteins in vivo.
31
19
F NMR can also be used as a sensitive probe of specific metabolic signals when a
fluorine atom can be incorporated into a small or large biomolecule as a reporter.
32,33
The
use of ligand-based fluorine NMR screening methods to rapidly identify biologically active
fluorinated organic compounds has demonstrated the usefulness of being able to detect a
19
F NMR signal in complex environments.
34,35
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 market containing at least one
fluorine atom.
34
Recently, a fluoride ligand has been incorporated into the axial position of
a Pt(IV) anticancer pro-drug to enhance stability and cytotoxicity.
36
Therefore, we believe
that incorporating fluorine substituents into Ru
II
arene complexes can be greatly beneficial
in probing their reactivity in complex mixtures, as well as a means of altering activity.
Throughout this study, we have used
19
F NMR to gather quantitative information, such as
kinetic parameters and percentage formation of the species present in solution, with mass
spectrometry used to support the assignment of species. This combined approach can
greatly simplify observing the binding preferences of Ru(II) arene in complex biological
mixtures. The purpose here, was not to develop a new bioactive ruthenium organometallic
compounds, but to explore a quantitative spectroscopic method for quickly and accurately
assigning the speciation of ruthenium organometallics in complex solutions.

3
Table 1: Complexes synthesized and studied in this work, isolated and used as the
hexafluorophosphate salts.
Incorporating the fluorine atoms into a bipyridyl ligand, which is stable to dissociation,
enabled at least two fluorines in chemically similar environments to be introduced to each
complex. We synthesised four fluorinated 2,2’-bipyridyl derivatives; 3,3’, 5,5’, 6,6’-difluoro
and 5,5’-trifluoromethyl. To counteract the electron withdrawing effect of the fluorinated
ligands on the metal, we also varied the arene ligand, including more electron-donating
substituted arenes by way of compensation. The full set of complexes reported is listed in
Table 1, and Figure S1.
Some of these complexes provide excellent sensitivity to the metal coordination
environment and dispersion of signals that have allowed us to resolve the surprising
number of different species present. The sensitivity we report suggests that this approach
could be developed for use in vivo and we have made attempts to test that. We also show
that the rates of formation of different 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.
RESULTS AND DISCUSSION
A number of literature preparations are available for the preparation of functionalised
bipyridines,
37,38
however, after achieving relatively poor yields using copper catalysed,
Ullman-type coupling reactions, the fluorinated bipyridines were synthesised using an
adapted palladium-catalysed homocoupling procedure in poly(ethylene) glycol (see
Experimental Section).
39
We found that all except the 4,4’-difluorobipyridyl derivative could
be made reliably in good yield; we have not pursued the 4,4’ derivative any further. The
synthesis of the ruthenium complexes [1] [12] was achieved by reacting the appropriate
ruthenium arene dimer with a stoichiometric amount of the chosen bipyridyl derivative.
These complexes were characterised by NMR, ESI-MS, elemental analysis and X-ray
diffraction.
Single crystals suitable for X-ray diffraction for complexes [1] [10] were grown via slow
diffusion of diethyl ether into a saturated solution of the complex in acetone. The X-ray
crystallographic data for all complexes are given in Tables S2-4. The novel complexes [1]-
[10] adopt a pseudo-octahedral piano stool configuration. A representative analysis of key
bond lengths and angles for the 5,5’-fluorinated bipyridine series is summarised in Table
S1. Complexes [1] and [2] have shorter Ru-Cl bond lengths than complexes [3] and [4]

4
which could provide a hint towards the lability of the chloride when other nucleophiles are
present, but these small differences cannot be used to inform solution based behavior.
Speciation in Aqueous Solutions
Before exposing the ruthenium complexes to biomolecules, they were monitored in
deuterated phosphate buffer, pD = 7.2 and 1% DMF which leads to an equilibrium
between chloro, aquo and phosphate adducts, Figure 1a. In order to be able to see as
many relevant species as possible, high concentrations of the complexes under study
were needed so a small amount of DMF was used, to enhance solubility. We have seen
no evidence of DMF coordinating under these conditions. For the fluorinated complexes
[1] - [8] and [10], three singlet peaks are observed in the
19
F NMR spectra, as represented
in Figure 1b. The behaviour of complex [9], the 6,6’-bipyridyl derivative, was significantly
different suggesting that the proximity of the fluorines to the metal impacted upon the
ligand exchange properties. For [11]-[12],
1
H NMR spectra were used to resolve the
speciation through the diagnostic metal-coordinated arene signals.
40
Figure 1: Figure 1a: The equilibria that exist when [Ru(h
6
-arene)(5,5’-difluorobipyridine)]
+
complexes are incubated in phosphate buffer. Figure 1b: Time course
19
F{
1
H} NMR
spectra of complex [4] incubated in 10mM deuterated phosphate buffer (2 mM Ru, pD =
7.2, 310 K). After 24 hr, 1 eq. of AgNO
3
was added to abstract the chloride ligand and
encourage formation of the aquo complex. Figure 1c:
19
F{
1
H} NMR spectra of complex [2]
incubated in D
2
O and buffered D
2
O of different phosphate concentration (2 mM Ru, pD =
7.2 (when buffered), 2 hr, 310 K). Figure 1d: Mass spectra recorded of the solution mixture
when complex [2] is incubated in D
2
O and buffered D
2
O, expected masses quoted in
Table S6.

5
With a number of titratable groups involved in these systems, examining the ligand
exchange behaviour of these complexes around conditions of pH relevant to biological
conditions requires strong buffering. We chose phosphate as a buffer also for its relevance
to biological conditions. However, it is clear that phosphate competes quite strongly as a
ligand to the metal , Figure 1c, and the presence of phosphate species have been
confirmed by mass spectrometry, Figure 1d and Table S6. Sadler et al. showed that the
structurally related [Ru(h
6
-arene)(en)Cl]
+
initially binds to the phosphate in the nucleobase
5-GMP before being displaced by the guanine N7.
41
To maintain a balance between
buffering strength and introducing a competing ligand a phosphate buffer concentration of
10 mM was chosen for all incubations, when the ruthenium concentration was 2 mM.
19
F resonances from the aquo and phosphate species are pH dependent and therefore the
pK
a
for the deprotonation of the bound aquo ligand can easily be measured. Figures 2a,
2b and 2c show representative data and calculated pK
a
values for the benzene complexes
with different bipyridyls (Complexes [1], [10] and [11]) and for the 5,5 difluorobipyridyl
complex with different arene ligands (Complexes [1] [4]). The observed differences in
pK
a
are entirely consistent with the changes in electron density at the metal due to the
subtleties of the electron withdrawing effects of fluorines on the bipyridyl ligand and the
electron donating effects of the different arenes.
42
In other words, the trend observed
going from complex [1] [4] shows that the increasing electron donating capability of the
h
6
-arene ligand to the ruthenium centre leads to a higher measured pK
a
value; an
increased electron density on the metal lowers its Lewis acidity. The electron withdrawing
capabilities of the trifluoromethyl group on complex [10] significantly increases the Lewis
acidity, reflected in the lowest pK
a
measured for any of these complexes. Interestingly,
pKa had to be measured using
1
H spectra because the
19
F resonances in this complex,
where the fluorine atoms are not directly on the bipyridyl ring, are not sensitive enough to
changes in coordination to accurately measure the pK
a
. Clearly this has implications for
the general use of
19
F on spectator ligands for reporting changes at the metal site.

Figures (10)
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TL;DR: Taken together, KP1019 and NKP-1339 are promising drug candidates, and especially the very limited side effects observed so far in clinical phase I trials seem to be a major advantage of this class of ruthenium drugs as compared to other chemotherapeutics and targeted anticancer compounds.
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Frequently Asked Questions (1)
Q1. What are the contributions in "Use of a fluorinated probe to quantitatively monitor amino acid binding preferences of ruthenium(ii) arene complexes" ?

In this paper, the binding preferences of Ru ( h-arene ) organometallic complexes were investigated using 1-dimensional 1H NMR spectra.