Tracking Reactions of Asymmetric Organo-Osmium Transfer Hydrogenation Catalysts in Cancer Cells

Article abstract of DOI:10.1002/anie.202016456

Most metallodrugs are prodrugs that can undergo ligand exchange and redox reactions in biological media. Here we have investigated the cellular stability of the anticancer complex [Os^II[(η⁶-p-cymene)(RR/SS-MePh-DPEN)] [1] (MePh-DPEN=tosyl-diphenylethylenediamine) which catalyses the enantioselective reduction of pyruvate to lactate in cells. The introduction of a bromide tag at an unreactive site on a phenyl substituent of Ph-DPEN allowed us to probe the fate of this ligand and Os in human cancer cells by a combination of X-ray fluorescence (XRF) elemental mapping and inductively coupled plasma-mass spectrometry (ICP-MS). The BrPh-DPEN ligand is readily displaced by reaction with endogenous thiols and translocated to the nucleus, whereas the Os fragment is exported from the cells. These data explain why the efficiency of catalysis is low, and suggests that it could be optimised by developing thiol resistant analogues. Moreover, this work also provides a new way for the delivery of ligands which are inactive when administered on their own.

Full text of DOI:10.1002/anie.202016456

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Anticancer Complexes
Hot Paper
Tracking Reactions of Asymmetric Organo-Osmium
Transfer Hydrogenation Catalysts in Cancer Cells
Elizabeth M. Bolitho, James P. C. Coverdale, Hannah E. Bridgewater,
Guy J. Clarkson, Paul D. Quinn,* Carlos Sanchez-Cano,* and Peter J. Sadler*
Angewandte
Chemie
6462
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Abstract: Most metallodrugs are prodrugs that can undergo
stable (over one month under normal atmospheric condi-
ligand exchange and redox reactions in biological media. Here
we have investigated the cellular stability of the anticancer
complex [OsII[(h6-p-cymene)(RR/SS-MePh-DPEN)] [1]
(MePh-DPEN = tosyl-diphenylethylenediamine) which catal-
yses the enantioselective reduction of pyruvate to lactate in
cells. The introduction of a bromide tag at an unreactive site on
a phenyl substituent of Ph-DPEN allowed us to probe the fate
of this ligand and Os in human cancer cells by a combination
of X-ray fluorescence (XRF) elemental mapping and induc-
tively coupled plasma-mass spectrometry (ICP-MS). The
BrPh-DPEN ligand is readily displaced by reaction with
endogenous thiols and translocated to the nucleus, whereas the
Os fragment is exported from the cells. These data explain why
the efficiency of catalysis is low, and suggests that it could be
optimised by developing thiol resistant analogues. Moreover,
this work also provides a new way for the delivery of ligands
which are inactive when administered on their own.
tions) than its industrially-used RuII analogue.[3] Furthermore,
once inside cells, and in presence of the non-toxic hydride
donor formate, this complex catalyses the enantioselective
reduction of pyruvate, an essential precursor in cell metab-
olism, to natural L-lactate or unnatural D-lactate, depending
on the chirality of the catalyst.[1f]
It can be assumed that the ability of such catalysts to cause
metabolic perturbations in cells requires the presence of
intact catalyst, which contributes to the antiproliferative
activity and selectivity of 1 towards a variety of human cancer
cell lines.[2e,4] Yet, its intracellular catalytic activity is most
likely marked by low turnover numbers (TON; previously
estimated to be ꢀ 13), which might suggest some degradation
of the complex inside cells.[1f] This might be expected to be
a common problem for synthetic metal catalysts, such as
organometallic complexes designed to work under well-
defined chemical conditions, including inert atmospheres
and in organic solvents.[1g,i,2c,5] In order to optimise the design
of synthetic intracellular catalysts, and increase their in cellulo
catalytic and biological efficiency, it is important to inves-
tigate their fate in cells.
Introduction
Transition metal catalysts have potential as therapeutic
agents to treat cancer and other diseases.[1] Such catalysts
might transform multiple substrate molecules in situ, includ-
ing exogenous prodrugs, diagnostic agents, and endogenous
metabolites,[1,2] whilst requiring only low concentrations to
achieve the desired activity. Therapeutic strategies based on
metal catalysts might help to overcome resistance to chemo-
therapy and reduce unwanted side effects,[1a,b] both of which
are of current clinical concern.
Previous work using ICP-MS experiments on fractionated
cancer cells treated with 1, showed a ca. 47% cytosolic
accumulation of the Os,[1f] suggesting that catalysis may take
place in the cytosol. Additionally, ca. 48% of intracellular Os
was present in the membrane/ particulate fraction (which
contains organelles and membrane proteins), which may also
implicate organelles (i.e. mitochondria or lysosomes) as
cellular targets.[1f] However, such studies did not provide
information on the intracellular stability of the complex.
To probe this, we have incorporated a halide substituent
on a sulfonylphenyl substituent in the chelated Ph-DPEN
ligand, so generating [OsII(h6-p-cym)(R1/2Ph-DPEN)], com-
plexes with R1(para) = Br (complexes R,R- and S,S-2), I
(R,R- and S,S-3), F (R,R-4), or R2(meta) = Br (S,S-5). A
combination of nanofocused synchrotron X-ray fluorescence
(XRF) and ICP-MS allows not only osmium but also the
chelated DPEN ligand to be tracked in cells using the halide
label. These experiments shed new light on the chemistry of
these organometallic catalysts in cells, and will aid the design
of next-generation catalytic drugs.
[OsII[(h6-p-cymene)(RR/SS-MePh-DPEN)] [1] (MePh-
DPEN = tosyl-diphenylethylene-diamine) is a chiral 16-elec-
tron organo-osmium(II) half-sandwich complex structurally
derived from the well-established Noyori RuII catalysts
(Figure 1a),[3] which shows high enantioselectivity and con-
version rates. For example, reduction of acetophenone is ca. 3-
fold more efficient (in turnover frequency, TOF) and more
[*] E. M. Bolitho, J. P. C. Coverdale, H. E. Bridgewater, G. J. Clarkson,
P. J. Sadler
Department of Chemistry, University of Warwick
Coventry, CV4 7AL (UK)
E-mail: P.J.Sadler@warwick.ac.uk
E. M. Bolitho, P. D. Quinn
I14 Imaging Beamline, Diamond Light Source
Oxford, OX11 0DE (UK)
Results and Discussion
We introduced halogen tags (R1 or R2) into chiral Ph-
diphenylethylene (Ph-DPEN) ligands by coupling phenyl-
sulfonyl chlorides carrying Br, I, or F in para or meta positions
to 1S,2S or 1R,2R diphenylethylenediamine. The new ligands
were then reacted with [Os(p-cymene)Cl2]2 dimer (synthes-
ised using a reported method)[6] to generate enantiomerically
pure organometallic complexes S,S- and R,R-2 (p-Br) and 3
(p-I), R,R-4 (p-F), and S,S-5 (m-Br), analogues of complex
1 (Figure 1a).
Halogenation of the phenyl group did not alter signifi-
cantly the structure of the complexes, as seen by comparison
of the X-ray crystal structures of complexes S,S-1, S,S-2 and
S,S-3 (Supporting Information, Table S1, Figures S7 and S8),
E-mail: paul.quinn@diamond.ac.uk
C. Sanchez-Cano
Center for Cooperative Research in Biomaterials (CIC biomaGUNE),
Basque Research and Technology Alliance (BRTA)
Paseo de Miramon 182, 20014 San Sebastiµn (Spain)
E-mail: csanchez@cicbiogamune.es
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.202016456.
ꢀ 2021 The Authors. Angewandte Chemie International Edition
published by Wiley-VCH GmbH. This is an open access article under
the terms of the Creative Commons Attribution License, which
permits use, distribution and reproduction in any medium, provided
the original work is properly cited.
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Figure 1. a) Structures of [Os(p-cymene)(R1/2Ph-DPEN)] complexes 1–5 (for R1 and R2 substituents, see Table 1). Only the (R,R) configuration is
shown for clarity. b) Accumulation of Os (ng/106 cells) in A2780 (human ovarian) cancer cells treated with 1”IC50 of R,R-1, S,S-2 and R,R-2
(310 K, 5% CO2) for 3, 6, 18 or 24 h (no recovery) or 24 h drug exposure followed by 24, 48 or 72 h recovery period (in complex-free media).
c) Transfer hydrogenation-modulated cellular viability of A549 (human lung) cancer cells treated with 0.5”IC50 of S,S-2 in the presence of formate
(0–2 mM). Statistical significance was calculated using a two-tailed t-test assuming unequal variances (Welch’s t-test), *p<0.05 and **p<0.01.
and led only to a moderate decrease in their hydrophobicity
(LogP 1.45 Æ 0.02 for 1, ca. 1 for 2, 3 and 5, and 0.30 Æ 0.03 for
4; Supporting Information, Table S2). Density Functional
Theory (DFT) calculations related such changes in hydro-
phobicity to differences in the effective charge distribution at
MCF7 and prostate PC3 cancer cells, while R,R-4 was more
potent against A2780 cells. Still, all of them were slightly less
potent than 1. (Figure 1d; Supporting Information, Table S7).
Similarly, they were also less toxic to zebrafish embryos, and
showed slightly improved biocompatibility (Supporting In-
formation, Table S20).[4] ICP-MS showed similar amounts of
Os in cells treated with equipotent concentrations of 1–3 after
24 h (dose of 1 ” IC50, Figure 1d; 0.25–1.5 ” IC50, Supporting
Information, Tables S10 and S11). Moreover, identical time-
dependent influx and efflux of osmium was observed in cells
treated with 1 ” IC50 of 1 and 2 (Figure 1b); reaching
maximum accumulation after ca. 6–8 h followed by a period
of concentration-independent efflux. Both complexes were
also taken up to some extent by cells using energy-dependent
mechanisms (i.e. endocytic pathways leading to lysosomal
deposition), as accumulation of intracellular osmium de-
creased at 277 K (Supporting Information, Table S12). Re-
duction in accumulation was significantly lower for cells
treated with 2.
À
the C X bonds due to the electronegativity of the halides
(F > Cl > Br > I, Supporting Information, Table S3, Fig-
ure S9). However, no significant effects were observed on the
charge at the Os center, p-cym arene, or N atoms of the R1/
2Ph-DPEN ligand (Supporting Information, Tables S3–6).
Equally, 2–5 maintained their ability to act as catalysts for
asymmetric transfer hydrogenation (ATH) reactions. They
catalysed the reduction of acetophenone to (S)- or (R)-1-
phenylethanol in presence of formic acid, achieving high
enantiomeric excesses and conversions (> 93%), with the Br
and I analogues (2, 3) showing slightly higher catalytic
efficiency when compared with 1 (TOF = 81 Æ 3, 87 Æ 5 and
63.6 Æ 0.6 hÀ1,[3] respectively; Table 1). This confirmed that
labelling Ph-DPEN ligands with halogen tags did not affect
significantly the chemical or catalytic properties of the new
complexes. Furthermore, 1H NMR and UV/Vis studies
showed that the halogenated analogues of 1 were stable over
24 h in DMSO or PBS.
The anticancer activity of R,R- and S,S-2 was improved
(cell survival reduced by 74%) by co-treatment with non-
toxic concentrations of hydride donor formate (Figure 1c;
Supporting Information, Figure S10), but not by acetate,
which cannot act as a hydride donor (Supporting Information,
Figure S11). The effect induced by formate was specific for
Enantiomers of 2 and 3 showed the same antiproliferative
activity in vitro against ovarian A2780, lung A549, breast
Table 1: Catalytic conversion (%), enantiomeric excess (%) and turnover frequency (TOF, hÀ1) for the reduction of acetophenone to (S)- or (R)-1-
phenylethanol for complexes 1–5 in the presence of 5:2 formic acid/ TEA azeotrope for 24 h at 310 K as determined by 1H NMR and chiral GC. Half-
maximal inhibitory concentrations (IC50/mM) of 1–5 towards A2780 (human ovarian) and A549 (human lung) cancer cells upon 24 h complex exposure
followed by a 72 h recovery period in fresh media. Osmium cellular accumulation in A2780 cells treated with 1”IC50 of 1–4 for 24 h (no recovery).
[a]
[c]
[c]
R1
R2
S/C
Conv. [%][a]
Config.[b]
ee [%][b]
TOFhÀ1
)
A2780 IC50
Os ng/106 A2780 cells[d]
A549 IC50
S,S-1[e]
R,R-1[e]
S,S-2
R,R-2
S,S-3
R,R-3
R,R-4[e]
S,S-5
CH3
CH3
Br
Br
I
I
F
H
H
H
H
H
H
H
H
Br
200
200
200
200
200
200
200
200
99
99
98
99
96
97
99
98
S
R
S
R
S
R
R
S
99
99
98
97
94
95
96
98
63.9Æ0.3
63.6Æ0.6
81Æ5
15.2Æ0.5
15.5Æ0.5
31Æ2
32Æ3
30Æ2
29Æ3
30Æ2
39Æ2
37Æ3
10Æ2
–
21.1Æ0.3
31Æ1
29.5Æ0.5
33Æ0.4
32Æ0.4
–
81Æ2
27.4Æ0.6
27.5Æ0.8
29Æ3
88Æ5
85Æ5
40Æ1
17Æ1
32Æ2
79Æ4
27Æ2
21.1Æ0.3
[a] Determined by 1H NMR. [b] Determined by chiral GC. [c] Determined using SRB colorimetric assay. [d] Determined by ICP-MS. [e] Previously
reported data for S,S-1, R,R-1 and R,R-4.[1f,3]
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+
cancer cells compared to normal healthy cells (Supporting
Information, Figure S12), and was not caused by an increase
in the intracellular levels of Os (p > 0.5; Supporting Informa-
tion, Table S13), or the disruption of membrane integrity
(Supporting Information, Table S8, Figure S13). However, as
previously observed for 1,[1f] the presence of 2 induced G1-
arrest (Supporting Information, Table S9). These results
confirm that halogen substitution does not affect significantly
the chemical, structural and catalytic properties of 2–5
compared to 1. The tagged complexes also show similar
biological properties to the parent complex. Complexes 2–5
appear to have the same mechanism of action than 1, being
capable of performing in-cell transfer hydrogenation. The
small decrease in their anticancer potency may be be caused
by reduced cellular accumulation, possibly related to lower
hydrophobicity.
The halogen tags used (i.e. F, Br and I) are present in most
biological organisms.[7] Bromine is reported to have an
important role in connective tissue formation,[8] but is found
in lower quantities than the other halogens in the body.[7a]
Bromine is also present only at low concentrations in
formulations for cell culture media and serum albumin.[7a,9]
Thus, low backgrounds were found when its accumulation and
distribution were determined in cellular samples that were
cultured in vitro (4.6 ng Br/ 106 cells). Furthermore, due to the
m/z = 79 or 81 (e.g. 40Ar39K+ or 31P16O3 for m/z = 79;
32S16O3 H+ or 40Ar40Ar1H+ for m/z = 81).[12] Furthermore,
1
nitric acid could not be used as the cell digestion matrix, as
it induces oxidation of bromine to molecular Br2. Instead,
pellets of A549 lung cancer cells treated with S,S-2 under
different conditions were digested at 353 K using an alkaline
solution of 25% w/v tetramethylammonium hydroxide
(TMAH). This reduced the loss of volatile analytes.[13] As
expected, intracellular levels of Br were much lower for
untreated cells than in cells exposed to S,S-2 (4.6 vs. 185 ng/106
cells after exposure to 30 mM of S,S-2 for 0 or 3, h,
respectively). The cells also accumulated Br and Os in
a different time-dependent manner (Figure 2a). The highest
levels of intracellular Os were reached after ca. 4–8 h, and the
levels of both Os and Br decreased upon recovery of cells in
drug-free media after 24 h exposure (Figure 2a; Supporting
Information, Table S14). However, there was a clear influx of
Br into cells for the whole duration of the exposure to the
complex (24 h). The Br tag in the complex was accumulated
> 9 ” more (on a molar basis) than Os after just 3 h exposure
to S,S-2 (Figure 2a; Supporting Information, Table S14).
ICP-MS measurements using isolated subcellular frac-
tions obtained from cells treated with S,S-2 also showed clear
differences in the cellular distribution of Br and Os (Fig-
ure 2b; Supporting Information, Tables S15–19). Osmium
was mostly (> 65%) in the fraction containing the mem-
branes and other organelles (i.e. mitochondria or lysosomes)
at all of the time points analysed (3–96 h). Lesser amounts of
Os were found in the cytoskeletal fraction, perhaps indicating
interactions between cationic osmium species and negatively-
charged microtubules, while very little was present in the
cytosol and nuclei of the treated cells (< 6%). In contrast,
while most Br was always present in the membrane and
organelle fraction (> 58%), much higher quantities of Br
compared to Os were found at all times in the cytosol and
nuclei of the treated cells (10–20%).
relatively high stability towards nucleophilic substitution of
[10]
À
the C Br bond in the BrPh-DPEN ligand in 2, release of
free bromide is unlikely to occur in the presence of biological
thiols in cellular conditions. Therefore, most of the detected
bromine should correspond to either complex 2 itself,
displaced or fragmented BrPh-DPEN ligand. Hence, we used
brominated complex 2 to probe the stability of this catalyst in
cells by determining the relative accumulation and local-
isation of Os and the Br tag, by detection of 189Os and 79Br by
ICP-MS, and by elemental mapping of Os and Br by detecting
nanofocused synchrotron X-ray fluorescence (XRF) emis-
sions of Os (L3-M5 = 8.9 keV) and Br (K-L3 = 11.9 keV).
Quantification of Br by ICP-MS was challenging due to
the high energy of its first ionisation potential (11.8 eV),[11]
and the presence of common polyatomic interferences with
ICP-MS experiments showed that not only the levels of
Os, but also Br decreased when cells were exposed to S,S-2 at
lower temperatures (277 K vs. 310 K, by ca. 48% and 75%,
respectively; Supporting Information, Figure S15). Lower Br-
Figure 2. a) Time-dependent accumulation of Os (189Os) and Br (79Br) in A549 (human lung) cancer cells treated with 1”IC50 (30 mM) of S,S-2 for
3, 8 18 or 24 h (no recovery) or 24 h followed by 24, 48 and 72 h recovery in drug-free medium, shown on a logarithmic scale for comparison.
b) Time-dependent accumulation of Os (189Os) and Br (79Br) in sub-cellular fractions (cytosol, membrane, nuclear and cytoskeletal) of A549 cells
treated with 1”IC50 (30 mM) of S,S-2 for various exposure times as quantified by ICP-MS.
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to-Os ratios found in cells upon inhibition of active transport
also suggested the presence of higher quantities of intact
complex (Supporting Information, Figure S16). Besides, ex-
cretion of Os and BrPh-DPEN seemed to occur through
different mechanisms. Inhibition of caveolae endocytic path-
ways using methyl-b-cyclodextrin reduced cellular uptake of
the complex (Supporting Information, Figure S17 and S18).
Yet, it did not alter the intracellular levels of Br, possibly by
inhibiting the cellular efflux of the free ligand. Moreover, Os
(but not Br) efflux was reduced when cells recovered in 20 mM
of verapamil after exposure to S,S-2 (Supporting Information,
Tables S18,19, Figure S19). Verapamil inhibits the ATP-
dependent efflux membrane pump Pgp (Permeability Glyco-
protein-1; a well-known pump involved in detoxification and
drug resistance).[14] Thus, uptake experiments suggested the
presence of intracellular degradation of the complex, fol-
lowed by differential cellular trafficking and efflux for Os and
Br-carrying fragments.
It is difficult to differentiate between membrane-bound
and internalized elements using 2D mapping techniques. Still,
concentration of large quantities of such elements in specific
organelles and cellular areas (i.e. nuclei, lysosomes, mito-
chondria, ER or other cytosolic organelles) can be easily
detected, providing vital information on their cellular distri-
bution. As such, the distribution of Os and Br in cancer cells
was further studied at sub-cellular spatial resolution (100 ”
100 nm2) by acquiring XRF elemental maps using nano-
focused synchrotron radiation at I14 (Diamond Light
Source). A549 lung cancer cells grown on silicon nitride
membranes were treated with various concentrations of S,S-2
(1–5 ” IC50 concentration) for 24 h, before being cryo-fixed
and freeze-dried for subsequent analysis under ambient
conditions (Figure 3). Natural intracellular levels of Br were
below the detection limit (Supporting Information, Fig-
ure S20). XRF emissions from Br or Os were not detected
in untreated cells (Supporting Information, Figure S21–24).
that intracellular Br was found in regions of the cytosol (71 Æ
9%, 60 Æ 7% and 52 Æ 7% at 1, 3 and 5 ” IC50, respectively),
but a significant amount of Br also reached the nuclei of
treated cells (29 Æ 9%, 40 Æ 7% and 48 Æ 7% at 1, 3 and 5 ”
IC50, respectively; Figure 3, Supporting Information, Ta-
ble S23). Remarkably, the percentage of Br in the nuclei
calculated from XRF maps (29 Æ 9%) was slightly higher than
that found when nuclear fractions were isolated from cells
treated under the same conditions (1 ” IC50) and analyzed
using ICP-MS (12 Æ 1%). XRF data are based on averaged
elemental determinations from between 3–7 individual cryo-
fixed, dried cells, in contrast to ICP-MS, performed on
digested cell fractions from a large population of cells. Also,
fractionation kits can introduce elemental leeching and cross-
contamination between fractions. Moreover, the data ob-
tained from XRF maps confirmed the trends found on our
previous ICP-MS experiments, and implied that intracellular
degradation of the complex was occurring. Nevertheless, the
Os and Br from the catalyst were found to co-localise
moderately in the cytoplasm (Pearsonꢀs Coefficient R =
0.24 Æ 0.11, 0.39 Æ 0.05 and 0.17 Æ 0.01 at 1, 3 and 5 ” IC50,
respectively; Supporting Information, Table S24). Thus, sug-
gesting that some of the complex should remain intact in
those areas after 24 h treatment, and could support transfer
hydrogenation catalysis in the cytosol of cancer cells. Fur-
thermore, both Os and Br appeared to accumulate in small
(0.65 Æ 0.21 mm2, 0.6 Æ 0.1 mm2 and 0.78 Æ 0.25 mm2 at 1, 3 and
5 ” IC50, respectively), cytoplasmic compartments of cells
when they were treated with S,S-2 (Figure 4a–c; Supporting
Information, Table S25, Figures S43–53). It is likely that these
are lysosomes or endosomes, since they are known to be
similar in size, and temperature-dependent accumulation
experiments had shown (at least) partial uptake of the Os
catalysts through endocytosis (or other active transport
mechanisms). Remarkably, Br/Os ratios were lower in those
areas than in the rest of the cell (4 Æ 2, 4 Æ 1 and 1.48 Æ 0.17 at
These control cells also maintained a normal “stretched out” 1, 3 and 5 ” IC50, respectively, Supporting Information,
morphology (as seen from S, P and K maps) typical of this cell
line,[17] and had clearly defined nuclei, areas with high
accumulation of Zn (Supporting Information, Figures S21–
24). On the contrary, XRF maps acquired from cells treated
with S,S-2 showed the presence of drug-induced morpholog-
ical changes, which were concentration-dependent (Figure 3;
Supporting Information, Figures S25–S38). For example, cells
became smaller in size and more rounded in shape when
treated with 1–3 ” IC50 of S,S-2. Instead, the use of higher
concentrations of the drug (5 ” IC50) led to cell swelling
Tables S23 and S25), suggesting the presence of higher
concentrations of intact complex.
Since the catalysts were at least partially taken up by
energy-dependent mechanisms and probably reached the
lysosomes, we investigated whether they are stable in acidic,
cysteine-rich environments typical of lysosomes (i.e. cysteine
proteases).[18,19] We incubated complexes 1 and 2 for 24 h at
pH 5.5 or 7, with 1 or 10 mM L-cysteine at 310 K. MS analysis
showed that the complexes alone remained intact at pH 5.5,
but in the presence of L-Cys they released their chelated
(Figure 3; Supporting Information, Table S21, Figures S39– MePh-DPEN or BrPh-DPEN ligands (Supporting Informa-
42) and nuclei with increased size and poorly-defined
perimeters due to a sparse intracellular distribution of Zn,
suggesting rupture of the nuclear membrane. This indicated
significant concentration-dependent cell damage caused by 2.
XRF elemental maps obtained from cells treated with S,S-
2 also showed that Os was located mostly in cytosolic regions
(from 75–85% for 1–3 ” IC50 treatment, to ca. 65% at 5 ” IC50;
tion, Table S26, Figures S54–S57). The fragment or fragments
carrying the Os center remain unidentified, as they could not
be isolated. It seems unlikely that dissociation of the Br-
labelled chelated ligand occurs in the culture medium used to
treat A549 cells since although we have shown that such
a dissociation can be induced by thiols, the level of thiols in
the medium is very low, with cysteine being present only as
Figure 3, Supporting Information, Table S22, Figures S25– oxidised cystine (0.2 mM) which does not react (Figure S58).
38). Equally, cells treated with the complex accumulated
more Br than Os (5, 7 and 2 ” more at 1, 3 and 5 ” IC50,
respectively; Supporting Information, Table S23). Most of
Cys34 in the foetal albumin present (ca. 30 mM), which is in
a cleft, would be expected to be inaccessible to such a bulky
organometallic complex, as found previously for
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Figure 3. XRF elemental maps acquired from cryo-fixed and freeze-dried A549 (human lung) cancer cells treated with 1–5”IC50 (30–150 mM) of
S,S-2 for 24 h showing the distribution of: Zn (green); (ii) K (blue) Os (orange) and Br (pink). Maps were collected using 100 nm step size and
0.1 s dwell time. Data were processed in PyMca toolkit developed by the ESRF,[15] and images generated in ImageJ.[16]
[(biphenyl)Ru(en)Cl]+.[20] Thus, degradation of the complexes
by thiols such as GSH seems likely to occur in lysosomes after
endocytosis. We tested this hypothesis by reducing cellular
levels of glutathione with low doses of L-buthionine sulfox-
imine (L-BSO), or inhibiting the activity of lysosomes in A549
cells with chloroquine diphosphate (which reduces activity of
lysosomal proteases and prevents endosome maturation).[21]
Remarkably, co-administration of L-BSO (5 mM; Figure S59)
or pre-incubation with chloroquine (150 mM, 2 h; Figure 5a)
led to a significant increase in the anticancer potency of 1 (by
36% or 49%, respectively) and 2 (by 25%, for chloroquine).
This may be due to increased accumulation of catalyst
(Figure 5b), but was not caused by a decrease in membrane
integrity in cells exposed to both chloroquine and the catalysts
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Figure 4. XRF elemental maps of Os and Br acquired from cryo-fixed and freeze-dried A549 (human lung) cancer cells treated with a) 1, b) 3 or
c) 5”IC50 of S,S-2 (30, 90 or 150 mM) for 24 h, showing Os-Br co-localisation in small vesicle-like areas. Maps were collected using 100 nm step
size and 0.1 s dwell time. The calibration bar is in pgmmÀ2. Data were processed using the PyMCa toolkit developed by the ESRF,[15] and images
generated in ImageJ.[16]
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Figure 5. a) Half-maximal inhibitory concentrations (IC50/mM) of R,R-1 and S,S-2 for A549 (human lung) cancer cells pre-incubated with 0 or
150 mM of chloroquine diphosphate for 2 h, followed by 24 h complex exposure and 72 h recovery period in fresh media. b) Accumulation of Os
(
189Os; ng/106 cells) in A549 cells pre-incubated with 0 or 150 mM chloroquine diphosphate for 2 h and treated with 1”IC50 of R,R-1 (21 mM) or
S,S-2 (30 mM) for 24 h. c) Molar Br-to-Os ratios (Br/Os) for A549 cells pre-incubated with 0 or 150 mM of chloroquine diphosphate for 2 h and
treated with 1”IC50 of S,S-2 (30 mM) for 24 h. d) Transfer hydrogenation-modulated cellular viability of human A549 cells pre-incubated with 0 or
150 mM chloroquine diphosphate for 2 h and treated with 1”IC50 of S,S-2 in the presence of formate (0–2 mM). Statistical significance was
calculated using a two-tailed t-test assuming unequal variances (Welch’s t-test), *p<0.05, **p<0.01 and ***p<0.001.
(Supporting Information, Table S27). It appeared that the
complexes were degraded less by chloroquine-treated cells, as
they accumulated higher quantities of Os and had lower Br/
Os ratios (Figure 5b,c, Supporting Information, Figure S60,
Table S28). Interestingly, chloroquine enhanced the antipro-
liferative response of 1 to formate, which supported the
presence of increased concentrations of intact catalyst inside
cells (Figure 5d, Supporting Information, Table S28).
Overall, these results provide new insights into the
behaviour of this family of asymmetric transfer hydrogena-
tion catalysts in cancer cells (Figure 6). The complexes are
internalised by cells via a combination of both passive and
active transport, reaching lysosomes, the cytosol and some
other organelles. Once inside, in the presence of a hydride
donor, intact catalysts facilitate the catalytic reduction of
pyruvate to lactate in the cytosol, altering the metabolism of
highly accumulated by cells, reaching even the cell nuclei
before they are excreted by vesicle-related exocytosis. This
raises questions about the role of the free ligands in the
activity of the complexes. However, degradation of 1 and its
analogues causes the loss of catalytic and biological activity of
the complexes, reducing their overall anticancer potential.
Furthermore, although RPh-DPEN ligands are readily in-
ternalised by cells (Supporting Information, Table S15), they
do not possess antiproliferative properties (IC50 > 150 mM,
Supporting Information, Table S7). Still, the interaction
observed between 1 and cellular thiols could provide a basis
for developing analogous therapeutic complexes designed for
the simultaneous delivery of active ligands and reactive
fragments of metal complexes inside cells.
cancer cells and inhibiting their proliferation. The catalysts Conclusion
also interact with intracellular thiols (e.g. cysteine-containing
peptides such as GSH and proteins). This leads to the release
of Os-containing fragments and intact (or fragmented) DPEN
chelating ligands, which are likely to be charged at physio-
logical pH.[22] The fragments generated during this degrada-
tion exhibit different cellular behaviour, which explained
differences observed between the accumulation of Os and Br.
Osmium-containing fragments are rapidly excreted from cells
via Pgp membrane pumps, while chelated ligands show much
longer in-cell lifetimes. As such, RPh-DPEN ligands are
We have labelled chelated ligands of anticancer organo-
osmium transfer hydrogenation catalysts with halogen atoms.
Such tags did not alter the chemical and biological properties
of the catalyst. Halogenation permits application of a range of
techniques to study the ligands of metal complexes in
biological environments, including: 19F NMR or MRI for
fluorinated compounds,[23] ICP-MS and XRF using bromine
or iodine tags,[9b, 24] or PET/SPECT imaging after radiolabel-
ling with 131I.[25] We probed the structure and spatial local-
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Figure 6. Mechanisms for the uptake of catalyst R,R-1 into cancer cells, its degradation and efflux. Created with BioRender.com.
isation of a brominated transfer hydrogenation catalyst inside
cells with ICP-MS and nanoscale synchrotron XRF mapping,
combined with cellular uptake and mechanistic studies. These
experiments showed that the catalyst was degraded in cancer
cells, probably through transport into acidic lysosomes
following by reaction with cellular thiols. The chelated Br-
ligand (or a Br-fragment), but not Os, is translocated into the
nucleus. Such reactions help to explain the low intracellular
TON estimated for these catalysts. This work demonstrates
the utility of halogen tags as probes for MS and X-ray based
techniques which elucidate reactions of organometallic anti-
cancer catalysts in cells. The next step for improving the
efficiency of the catalysts might be to tether the chelated
ligand to the arene ring, a strategy which has been used
effectively in chemical systems.[26] On the other hand, such
ligand release reactions might be used to deliver drugs which
would otherwise be poorly taken up into cells and be inactive
Acknowledgements
We thank the Engineering and Physical Sciences Research
Council (EPSRC grant no. EP/P030572/1), and Warwick
Collaborative Postgraduate Research Scholarship and Dia-
mond Light Source (DLS, Oxford) for a studentship for
E.M.B. C.S.C. thanks Gipuzkoa Foru Aldundia (Gipuzkoa
Fellows program; grant number 2019-FELL-000018-01/62/
2019) for financial support. This work was performed under
the Maria de Maeztu Units of Excellence Programme— Grant
No. MDM-2017-0720 Ministry of Science, Innovation and
Universities. We thank I. Bagley for assistance with zebrafish
experiments, J. I. Song with cell experiments, S. E. Bakker and
I. Hands-Portsman (Advanced Bioimaging RTP) with
plunge-freezing, A. Catherwood and J. Tod with the freeze-
dryer, J. Barrios-Rivera and S. Forshaw with GC-FID, J.
Parker and F. Cacho-Nerin at the I14 beamline, L. Song and J.
Morrey with mass spectrometry, I. Prokes with NMR
spectroscopy, and M. Wills for stimulating discussions. All
synchrotron work was performed at the I14 Beamline (DLS,
Oxford) under experiment number sp20552-1.
if administered separately.
Data Availability: The data that support the findings in this study
are available in the Warwick Research Archive Portal (WRAP)
repository, http://wrap.warwick.ac.uk/147754/.
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Conflict of interest
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The authors declare no conflict of interest.
Keywords: anticancer catalysts ·bioorganometallic chemistry ·
X-ray fluorescence ·organo-osmium complexes ·
transfer hydrogenation
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Manuscript received: December 11, 2020
Revised manuscript received: January 16, 2020
Version of record online: February 15, 2021
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