Folates are potential ligands for ruthenium compounds in vivo

Article abstract of DOI:10.1039/c4dt00081a

Under physiologically relevant conditions, cis-bis(2,2′-bipyridine) dichlororuthenium(ii), [cis-Ru(2,2′-bipy)₂Cl₂] was observed to bind to folic acid via replacement of the two chloride ligands. This binding was shown to be pH depend

Full text of DOI:10.1039/c4dt00081a

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Folates are potential ligands for ruthenium
compounds in vivo†
Cite this: Dalton Trans., 2014, 43,
8158
Tom G. Scrase, Simon M. Page, Paul D. Barker* and Sally R. Boss*
Received 9th January 2014,
Accepted 19th March 2014
DOI: 10.1039/c4dt00081a
www.rsc.org/dalton
Under physiologically relevant conditions, cis-bis(2,2’-bipyridine)-
dichlororuthenium(II), [cis-Ru(2,2’-bipy)2Cl2] was observed to bind
to folic acid via replacement of the two chloride ligands. This
binding was shown to be pH dependent and afforded diastereo-
mers, the structures of which were determined by 1- and 2D NMR
spectroscopic techniques. We propose that when studying the
cytotoxicity of labile ruthenium complexes in cells, folate coordi-
nation should be considered.
The orthogonal chemistry of the complexes of heavier tran-
sition-metals has received much attention with respect to their
potential to yield novel drug candidates.1,2 Such complexes
offer a possible spectrum of activity which extends beyond
organic small molecules, simply because of the propensity for
metals to form strong coordinate bonds to Lewis bases.3
Recent advances in the field have seen ruthenium-based com-
pounds emerging as some of the most promising drug
candidates.4–6 Two such compounds, KP1019 and NAMI-A,
have now entered clinical trials, passing phase 1 stages.7–9
However, their precise mode of action and their preferred
in vivo target or targets have yet to be unequivocally established
and this is a barrier to their further development.10–12
The potential for DNA binding of such compounds has
been shown in vitro,13–15 however, there is also evidence for
binding to proteins as well as DNA.16 Indeed, the cytotoxicity
of ruthenium complexes may be as a result of binding to mul-
tiple targets,12 including small molecules, such as metabolites
and cofactors, which have yet to be considered. Without a
fuller understanding of the interaction of ruthenium com-
pounds with all biomolecules, large and small, a strategic
approach to improving metal-based drugs will remain challen-
ging. In this context folates are relevant biomolecules; whilst
Fig. 1 The structure of folic acid with the atoms that could contribute
to chelating sites numbered. The oxygen at position 4 can be considered
as either a carbonyl or iminol depending on which tautomeric state is
relevant (inset).
they are not in high concentration they are ubiquitous cofac-
tors in vivo and central to metabolite biosynthesis. Hence,
folates are likely to be encountered by any metal complex
administered. With several Lewis basic functional groups avail-
able, folates offer a range of potential binding motifs to a
metal (see Fig. 1).
We have investigated the products formed between
[cis-Ru(2,2′-bipy)2Cl2] and folic acid in vitro. Whilst ruthenium
compounds of clinical interest have one or more labile, mono-
dentate ligand, we expect the interaction of folates with these
complexes to yield numerous products such that detailed
structural characterisation would be precluded. With only two
labile ligands and relatively limited conformational freedom,
[cis-Ru(2,2′-bipy)2Cl2] offers a ruthenium centre that can
accommodate mono or bidentate ligands whilst retaining the
chelating bipyridine ligands and hence provides an ideal
centre to explore the reaction with folate, including compe-
tition with monodentate ligands. The potential for polydentate
binding allows for tight, biologically irreversible chelate
formation.
The University of Cambridge Chemical Laboratory, Lensfield Road, Cambridge, CB2
1EW, UK. E-mail: pdb30@cam.ac.uk, srb39@cam.ac.uk; Fax: +44 (0)1223 336362;
A solution of 5.0 mM [cis-Ru(2,2′-bipy)2Cl2·2H2O] and
5.0 mM folate was stirred at 37 °C in phosphate buffered
saline. MS of the reaction mixture shows no sign of free folic
acid with all major ruthenium containing signals correspond
Tel: +44 (0)1223 336300
†Electronic supplementary information (ESI) available: Full experimental pro-
cedures, compound characterisation and copies of NMR and MS spectra. See
DOI: 10.1039/c4dt00081a
8158 | Dalton Trans., 2014, 43, 8158–8161
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to folate bound species within a day (m/z = 427.6; [cis-Ru(2,2′- and the intensity of these signals indicate that the diastereo-
bipy)2(folic acid)]2+, 854.4; [cis-Ru(2,2′-bipy)2(folic acid − H+)]+ mers are present in a ∼2 : 1 ratio (ΛS/ΔR : ΛR/ΔS). In both
following the deprotonation of the folic acid). Attempts to cases the N10 signals show coupling to two distinct, protic
isolate the product of this reaction for further analysis were environments on the adjacent C9 atom (one diastereomer
complicated by the high salt content of the buffer. In order to showing a doublet of doublets and the other a triplet). This is
investigate the folate bound species more fully, [cis-Ru(2,2′- in contrast to a broad singlet observed at 6.90 ppm for the N10
bipy)2Cl2·2H2O] was reacted with stoichiometric folic acid at environment of free folic acid. The constrained environment at
65 °C overnight in aqueous solution with no added salt and C9, leading to the loss of degeneracy of the two C9 protons
then the product isolated as the hexafluorophosphate salt in (3.20, 3.87 ppm for ΛS/ΔR isomers; 3.23, 3.90 ppm for ΛR/ΔS
59% yield. The MS data of the compound as synthesised via this isomers), suggests this methylene group is now part of the che-
route was consistent with the folate bound species formed lating ring. These shifts also contrast to those of free folic acid
under more physiological conditions. The reaction in water can where a singlet is observed at 4.48 ppm for the C9 protons.
easily be monitored by ESI-MS revealing ruthenium species such Further key evidence comes from the NOEs highlighted in
as [cis-Ru(2,2′-bipy)2(H2O)(OH)]+ and [cis-Ru(2,2′-bipy)2(H2O)Cl]+ Fig. 2 including the NOEs from bipyridine environments 6 and
(m/z = 449.0, 467.0 respectively) immediately upon solvation. 6′ to folate environments N10 and 12/16. These assignments
These substitutions may precede folate binding; however, it is are consistent with all other spectroscopic assignments made
worth noting that the unsubstituted [cis-Ru(2,2′-bipy)2Cl2] is a (see ESI†). This motif of N,N chelation is also consistent with
neutral species and is less easily detected by this method.
The structure of the isolated product was determined by folate metalation with cobalt(II) and nickel(II).17,18
1- and 2D, 13C and 1H NMR spectroscopic techniques (APT,
The reaction was repeated at 65 °C at pH 2.5 and 6.0 (both
what has been described to be the thermodynamic product of
HSQC, HMBC, COSY and NOESY.) The 13C NMR spectrum was citrate buffer) and at pH 9.9 (CAPS buffer) and the species
collected on a 500 MHz spectrometer and the 1H spectrum formed followed by ESI-MS in order to follow the formation of
and all 2D spectra on a 700 MHz spectrometer (see ESI†) and the ruthenium species over a range of pH. These three points
the product further characterised by ESI-MS, UV-Vis spectro- were chosen to reflect the possible protonation states of the
scopy and elemental analysis.‡ The NMR spectra are consist- folic acid i.e. predominantly protonated at the pterin moiety
ent with a bidentate binding motif of diastereomers with and therefore positively charged, neutral, and the carboxylate
coordination via sites N5 and N10 (see Fig. 2), forming a anion, respectively.19,20 Coordinate bond formation between
5 membered ring containing two new ruthenium–nitrogen the ruthenium complex and folic acid was observed at pH 6.0
coordination bonds. The main evidence supporting such and 9.9, however, at pH 2.5 no binding was evident, even after
N,N coordination includes the observation of two distinguish- 3 days of heating. This shows that significant protonation of
able species in the NMR spectra, which we assign to the two folate atom N5 inhibits binding to the ruthenium centre, con-
pairs of diastereomers. The largest shift difference between sistent with the nitrogen lone pair at this site being integral to
the two diastereomers is observed at N10 (6.08 and 6.23 ppm) chelation.
The proposed binding motif through the N5 and N10 nitro-
gens was investigated further by reaction of 10-formyl folic
acid21 with [cis-Ru(2,2′-bipy)2Cl2·2H2O]. No adduct formation
was observed at 37 °C, so the reaction was repeated at 65 °C
overnight in aqueous solution but yielded only starting
materials. This lends further weight to the importance of N10
in adduct formation.
Ruthenium(II) complexes of flavins, pterins and alloxazines
have been synthesised and studied from an electrochemical
point of view.22–24 Whilst an adduct between [cis-Ru(2,2′-
bipy)2]2+ and folic acid has been reported,23 a limited analysis
of the product led to the conclusion that folic acid had che-
lated to the metal through the O4 and N5 in a motif analogous
to flavin coordination. Such binding would produce a single
pair of enantiomers (identical by NMR spectroscopy). On
replication of the experimental conditions outlined,23 our sub-
sequent NMR spectral analysis of the complexes isolated once
Fig. 2 Section of the homonuclear NOESY spectrum illustrating the
two clearly distinguishable diastereomers. In one binding mode (ΛS/ΔR) again supports chelation to the ruthenium centre via N5 and
folate environment 10 shows NOEs to 2,2’-bipyridine environments 6
and 6’. In the other binding mode (ΛR/ΔS) an NOE is only observed
between folate environment 10 and 2,2’-bipyridine 6’. The unlabelled
cross peaks are due to folate environments 12/16 (5.90 ppm), 18
N10 with the same diastereomeric products being observed.
Interestingly, these more energetic conditions appeared to
favour the ΛS/ΔR isomers further as the NMR spectra
suggested formation of these in a ∼4 : 1 ratio relative to the
(8.15 ppm) and 19 (4.34 ppm). Residual water (3.33 ppm) is also evident.
See Tables S-3, S-4 and S-5† for full assignment of these data.
ΛR/ΔS isomers.
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Analysis of the isolated, synthetic product formed at 65 °C cellular balance of this cofactor. Folate metabolism has long
allowed us to interpret the NMR spectra of the more physio- been recognised as a key target for cancer therapy and folate
logically relevant mixture. Integration of the N10 proton uptake into tumour cells is significantly stimulated.33–35
signals suggest ∼2 : 3 ratio of ΛS/ΔR : ΛR/ΔS and, by compari- Understanding how ruthenium complexes interact with the
son to the N10 signal of free folate present, ∼90% conversion folate pool may be of significance.
of [cis-Ru(2,2′-bipy)2Cl2] to the folate coordinated complex. The
Nonetheless, the tight binding and slow exchange rates of
physiological relevance of such reactivity was explored further precious metal complexes are attractive properties to incorpor-
by following a solution of 4.8 mM [cis-Ru(2,2′-bipy)2Cl2·2H2O] ate into a medicinal compound in general. To selectively
and 4.8 mM dihydrofolate (DHF) at 37 °C under aqueous con- harness the potential of organometallic complexes as metallo-
ditions by ESI-MS. DHF was observed to bind within 2 days drugs, suitable targets need to be established and rationalised
(m/z = 428.7; [cis-Ru(2,2′-bipy)2(DHF)]2+) followed by the for- and a selective delivery strategy must be adopted to enable
mation and binding of folic acid in solution (m/z = 427.7; specific targeting to molecules of choice.
[cis-Ru(2,2′-bipy)2(folic acid)]2+, m/z = 442.1; [folic acid + H+]+)
after a further 24 hours.
We thank the EPSRC and Cancer Research UK for funding
the studentships of Tom Scrase and Simon Page, respectively,
The total concentration of folate species in cells is low, and and Michael O’Neill for critical reading of the manuscript.
of these, >90% are polyglutamylated at the glutamate end of
the molecule.25 As with the 10-formyl folic acid complex above,
not all of these folate species have available lone pairs at N5
Notes and references
and N10 for coordination. Nonetheless, the total ruthenium
content of cultured cells26 can be >40 fold higher than folate in
‡λmax = 470 nm. Theoretical: C = 40.91%, H = 3.08%, N = 13.46%. Results: C =
41.06%, H = 3.20%, N = 13.36% (2.0806 mg); C = 40.92%, H = 3.15%, N =
molar terms and hence the potential for complexation and the
long lifetime of the resulting species will interfere with enzyme
binding and the one-carbon carrying role of folates in cells.
The timescale of our results is consistent with the slow
ligand exchange rates that are typical of ruthenium-based com-
pounds27 including those that are being investigated for their
cytotoxic properties.28–30 Indeed it is these slow ligand-
exchange rates that are likely to be more important than the
absolute affinity of folate for the metal centre.
13.25% (1.6554 mg). m/z = 427.7; [cis-Ru(2,2′-bipy)2(folic acid)]2+ m/z = 854.3;
[cis-Ru(2,2′-bipy)2(folic acid − H+)]+.
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